WO2017011452A1 - Échafaudages électroactifs et procédés d'utilisation d'échafaudages électroactifs - Google Patents

Échafaudages électroactifs et procédés d'utilisation d'échafaudages électroactifs Download PDF

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WO2017011452A1
WO2017011452A1 PCT/US2016/041889 US2016041889W WO2017011452A1 WO 2017011452 A1 WO2017011452 A1 WO 2017011452A1 US 2016041889 W US2016041889 W US 2016041889W WO 2017011452 A1 WO2017011452 A1 WO 2017011452A1
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conductive
foam
silk
polymer
foams
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John Hardy
Christine E. Schmidt
David L. Kaplan
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Tufts University
University of Florida Research Foundation Inc
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University of Florida Research Foundation Inc
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • C—CHEMISTRY; METALLURGY
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1353—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)
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    • C12N2529/00—Culture process characterised by the use of electromagnetic stimulation
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
    • C—CHEMISTRY; METALLURGY
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    • C12N2535/00—Supports or coatings for cell culture characterised by topography

Definitions

  • Bone tissues are hierarchically structured composite materials composed of both soft and hard matter (i.e. cell-rich vascularized soft tissue, and collagen-/hydroxyapatite-rich hard tissue). Bone conditions and disorders that require surgical intervention motivate
  • Embodiments of the present disclosure provide for conductive foams (e.g., silk foams, polymer foams) methods of making the conductive foam, method of using the conductive foam, and the like.
  • conductive foams e.g., silk foams, polymer foams
  • An embodiment of the present disclosure includes a method of differentiation of human mesenchymal stem cells, among others, that includes: providing a conductive foam with an interpenetrating polymer network on the surface of the foam, wherein the
  • interpenetrating polymer network is made from a doped conductive polymer that is either a self-doped conducting polymer or a conducting polymer and a dopant; introducing human mesenchymal stem cells to the conductive foam, wherein the conductive foam and the human mesenchymal stem cells are cultured in a osteogenic medium; and periodically providing electrical stimulation to the human mesenchymal stem cells to cause differentiation of human mesenchymal stem cells towards osteogenic outcomes.
  • the method further includes providing increased ALP activity, increased collagen deposition, increased Ca 2+ deposition, or a combination thereof, on the foams relative to not periodically providing electrical stimulation.
  • An embodiment of the present disclosure includes a structure, among others, that includes: a conductive foam having an interpenetrating polymer network on the surface of the foam, wherein the interpenetrating polymer network is made from a doped conductive polymer that is either a self-doped conducting polymer or a conducting polymer and a dopant, wherein human mesenchymal stem cells are disposed within the conductive foam.
  • Figures 1 A-B demonstrate physicochemical analysis of the tissue scaffolds.
  • Fig. 1 A is an SEM image of non-conductive silk foam with inset photograph of the bulk foam.
  • Fig. IB is an SEM image of conductive silk foam, with inset photograph of the bulk foam, and the structure of the self-doped CP composed of pyrrole and 2-hydroxy-5-sulfonic aniline overlaid. Scale bars represent 400 ⁇ , and the bulk foams were 4 mm in diameter and height.
  • Figs. 1C and ID are XPS and FTIR spectra, respectively; grey lines represent spectra of non- conductive silk foams and black lines represent spectra of conductive silk foams.
  • Figures 2A-F are biochemical analyses.
  • Figure 2A illustrates in vitro degradation assay: white bars, silk foam without enzyme; light grey bars, silk foam with enzyme; dark grey bars, conductive silk foam without enzyme; black bars, conductive silk foam with enzyme.
  • Figure 2B illustrates HMSC viability after incubation with ethanol (15% v/v, toxic control) or different concentrations of CP.
  • Figures 2C-F illustrate quantitative studies of cell culture experiments: light grey bars, silk foam; dark grey bars, conductive silk foam without electrical stimulation; black bars, conductive silk foam with electrical stimulation.
  • Figures 3 A-R show histological analysis of the scaffolds at various points in time.
  • Hematoxylin and eosin (H&E) staining of sections of non-conductive scaffolds results in characteristic blue staining of cell nuclei, and characteristic pink staining of intracellular and extracellular proteins (e.g., actin or silk, respectively); Alizarin staining results in
  • Figures 3 A to 3F are non-conductive silk foams: Fig. 3 A illustrates 10 days, H&E, Fig. 3B illustrates 10 days, Alizarin, Fig. 3C illustrates 20 days, H&E, Fig. 3D illustrates 20 days, Alizarin, Fig. 3E illustrates 30 days, H&E, Fig. 3F illustrates 30 days, Alizarin.
  • Figures 3G to 3L are conductive silk foams without electrical stimulation: Fig. 3G illustrates 10 days, H&E, Fig. 3H illustrates 10 days, Alizarin, Fig. 31 illustrates 20 days, H&E, Fig.
  • FIG. 3J illustrates 20 days, Alizarin
  • Fig. 3K illustrates 30 days
  • Alizarin Figures 3M to Fig. 3R are conductive silk foams with electrical stimulation: Fig. 3M illustrates 10 days, H&E, Fig. 3N illustrates 10 days, Alizarin, Fig. 30 illustrates 20 days, H&E, Fig. 3P illustrates 20 days, Alizarin, Fig. 3Q illustrates 30 days, H&E, and Fig. 3R illustrates 30 days, Alizarin. Scale bars represent 100 ⁇ .
  • Figure 4 shows high magnification SEM image of the smooth underside of the foams in contact with the petri dish during foam preparation (foams are subsequently removed from the petri dish template during salt leaching).
  • the left side of the image is of non-conductive silk foam.
  • the right side of the image is of conductive silk foam clearly shows the presence of CP-based nanoparticles on the surface of the silk.
  • Scale bar represents 1 ⁇ .
  • Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of organic chemistry, biochemistry, microbiology, molecular biology,
  • Embodiments of the present disclosure provide for conductive foams (e.g., silk foams, polymer foams), methods of making the conductive foam, methods of using the conductive foam, and the like.
  • a cell such as a human mesenchymal stem cell can be incubated with the conductive silk foam and cultured in an osteogenic medium so that the stem cells differentiate towards osteogenic outcomes.
  • embodiments of the disclosure provide for methods of differentiation of human mesenchymal stem cells.
  • An embodiment of the present disclosure includes introducing human mesenchymal stem cells to the conductive foam (e.g., conductive silk foam), where the conductive foam and the human mesenchymal stem cells are cultured in an osteogenic medium.
  • the conductive foam has electroactive characteristics so that an electrical stimulation can be periodically applied to the conductive foam.
  • electrical stimulation can be periodically applied to the human mesenchymal stem cells to cause differentiation of human mesenchymal stem cells towards osteogenic outcomes.
  • application of electrical stimulation to the conductive silk foam increases ALP activity, increased collagen formation, and/or increased Ca 2+ deposition on the foams of the nonwoven mat, which can lead to formation of calcified bone-like extracellular matrix.
  • electrical stimulation of the conductive silk foam in the presence of human mesenchymal stem cells in the osteogenic medium shows increased differentiation towards osteogenic outcomes as compared to conductive silk foam without electrical stimulation and other types of materials.
  • the interpenetrating polymer network is formed from a conducting polymer (e.g., polypyrrole) and a dopant (e.g., 2-hydroxy-5-sulfonic aniline).
  • the conducting polymer can include either a self-doped conducting polymer or a conducting polymer and a dopant.
  • the interpenetrating polymer network is disposed on the surface of the conductive foams.
  • the amount of the conducting polymer and dopant disposed on the conductive foams can be about 1 to 100% by mass, where the range includes each 1% increment (e.g., 1 to 10%, about 50 to 80%, about 20 to 60%, and the like).
  • the conductive foam is a conductive silk foam that has a porous scaffold of silk protein having interpenetrating polymer networks.
  • the silk foams can include fibroin or related proteins.
  • the silk can be natural silks from any species, including but not limited to: silk worms, spiders, lacewings, caddisfly larvae, bees; or recombinantly/genetically engineered proteins inspired by silks; or chemically produced peptides/proteins/polymers inspired by silks.
  • fibroin includes fibroin obtained from a solution containing a dissolved silk (typically silkworm silk).
  • the silkworm silk protein may be obtained, for example, from B.mori, and the spider silk may be obtained from N. clavipes.
  • the silk can be obtained from a solution containing a genetically engineered silk, produced by bacteria, yeast, mammalian cells, transgenic animals or transgenic plants.
  • Silk solutions can be prepared by any conventional method known to one skilled in the art, and one approach is described in detail in the Example.
  • the conductive foam has usable mechanical properties as well as pore structure, where the one or more of the pores can be interconnected with or among one another.
  • the conductive foam can have a length of about 1 to 1000 millimeters and a width of about 1 micrometers to 1000 millimeters.
  • the density of the conductive foam can be about 0,01 and 10 g per cm 3 .
  • the porosity of the conductive foam can be about 3 and 99%, and the pores are typically interconnected and have pore sizes of 10 to 1000 ⁇ , where the diameter may be the same throughout the pore or vary in diameter.
  • the conductive foam can be a conductive polymer foam such as a synthetic polymer, a natural polymer, or a combination thereof.
  • the synthetic polymer can include polycaprolactone, polyesters, polyamides, polycaprolactone (PCL), poly-L-lactic acid (PLLA), poly lactic-co-glycolic acid (PLGA), and combinations thereof.
  • the natural polymer can be proteins, polysaccharides, lignins, polyalanine, oligoalanine, collagen, cellulose, chitin, chitosan, and a combination thereof.
  • the conductive foam can include a mixture of different types of polymers and/or silks (e.g., a portion of proteins such as silks and polysaccharides such as hyaluronic acid).
  • the interpenetrating polymer network can be formed from the conducting polymer and the dopant.
  • the conducting polymers include polypyrrole, polyaniline, polythiophene, poly(3,4-ethylenedioxythiophene), poly fluorenes, polyphenylenes, polypyrenes, polyazulenes, polynapthalenes, polyindoles, polyazepines, poly(p-phenylene sulfide)s, poly(p-phenylene vinylene)s, and polyfurans.
  • biodegradable versions in which there are block of conducting units within a polymer chain containing biodegradable bonds (e.g. esters and amides), that can also be used as the conducting polymer.
  • the dopant can be a polymer that has the opposite charge to the conducting polymer, and can have a low molecular weight (e.g., chlorine ions, tosylate ions, and the like) or high molecular weight (e.g., collagen, hyaluronic acid, and the like).
  • the dopant can be chemically linked to the backbone of the polymer (i.e., self- doped) potentially as a monomer (e.g. 2-hydroxy-5-sulfonic aniline).
  • the conducting polymer and the dopant can be disposed on the silk foams by incubating the foams with conducting polymer, dopant, and an agent capable of polymerizing the monomer(s) (e.g., an oxidizing agent such as ferric chloride, a light source or suitable electrochemistry apparatus) for an appropriate time period (e.g., about 1 to 36 hours or about 24 hours). After incubation, the residual materials can be washed away and the foams having interpenetrating polymer networks are formed. Additional details are provided in Example 1.
  • an agent capable of polymerizing the monomer(s) e.g., an oxidizing agent such as ferric chloride, a light source or suitable electrochemistry apparatus
  • the osteogenic medium is based on standard cell culture medium with the optional addition of other components such as serum, non-essential amino acids, bone morphogenetic protein 2 (BMP-2), dexamethasone, ⁇ -glycerophosphate, ascorbic acid, ascorbic acid-2-phosphate, heparin, retinoic acid, and 1,25-dihydroxycholecalciferol (for example: high glucose Dulbecco' s Modified Eagle Medium (DMEM, 425 mL); fetal bovine serum (50 mL); antibiotic-antimycotic (5 mL); non-essential amino acids (5 mL),
  • BMP-2 bone morphogenetic protein 2
  • dexamethasone for example: high glucose Dulbecco' s Modified Eagle Medium (DMEM, 425 mL); fetal bovine serum (50 mL); antibiotic-antimycotic (5 mL); non-essential amino acids (5 mL),
  • dexamethasone 100 nM
  • ⁇ -glycerol phosphate 10 mM
  • ascorbic acid 50 ⁇
  • the volume of medium used should be in line with the recommended guidelines of the manufacturer of the cell culture dishes.
  • Electrical stimulation can include direct contact of the material with a power source via a wire, wireless energy transfer, magnetic force, and the like.
  • the term "periodically” refers to applying the electrical stimulation at established time frames that may be at regular or irregular time intervals on the time frames of seconds, hours, days, weeks, or months (e.g., about 1 s to 2 months, about 1 hour to 1 day, about 1 day to 1 month, or other the like) depending upon the specific circumstances.
  • the impulses of the electrical stimulation can last on the time frame of seconds, hours, or days (e.g., about 1 second to 1 day, about 10 seconds to 1 hour, about 1 minute to 12 hours, about 1 hour to 1 day, or the like) depending upon the specific circumstances.
  • the electrical stimulation can be in the range of millivolts to volts (e.g., about 10 mV to 10 volts, about 1 mV to 100 mV, or the like).
  • the time frame, duration of electrical stimulation, and intensity of the electrical stimulation can be designed based on particular circumstances and requirements of a specific situation.
  • embodiments of the present disclosure provide for a conductive foam, where the foam includes interpenetrating polymer networks.
  • human mesenchymal stem cells e.g., on collagen-1 coated substrates
  • the differentiated products of the stem cells are disposed within the conductive foam.
  • the conductive foam includes ALP, collagen, and/or Ca 2+ , which can be deposited on the foams.
  • the conductive foam can include one or more agents (e.g., a chemical or biological agent), where the agent can be disposed indirectly or directly on the conductive foam.
  • agents e.g., a chemical or biological agent
  • the agent can include a stem cell such as a human mesenchymal stem cell.
  • an additional agent that can be disposed on the conductive foam can include, but is not limited to, a drug, a therapeutic agent, a radiological agent, a small molecule drug, a biological agent (e.g., polypeptides (e.g., proteins such as, but not limited to, antibodies (monoclonal or polyclonal)), antigens, nucleic acids (both monomeric and oligomeric), polysaccharides, haptens, sugars, fatty acids, steroids, purines, pyrimidines, ligands, and aptamers) and combinations thereof, that can be used to image, detect, study, monitor, evaluate, and the like, the differentiation of the stem cells.
  • the agent is included in an effective amount to accomplish its purpose (e.g., ALP production and/or Ca 2+ production), where such factors to accomplish the purpose are well known in the medical arts.
  • the agent can be bound to the conductive silk foam by a physical, biological, biochemical, and/or chemical association directly or indirectly by a suitable means.
  • bound can include, but is not limited to, chemically bonded (e.g., covalently or ionically), biologically bonded, biochemically bonded, and/or otherwise associated with the electroactive supramolecular polymeric assembly.
  • being bound can include, but is not limited to, a covalent bond, a non-covalent bond, an ionic bond, a chelated bond, as well as being bound through interactions such as, but not limited to, hydrophobic interactions, hydrophilic interactions, charge-charge interactions, ⁇ - ⁇ stacking interactions, combinations thereof, and like interactions.
  • cell-conductive silk foam interactions could be controlled through the inclusion of cell-adhesive peptides (e.g., RGD, YIGSR, KQAGDV, KHIFSDDSSE, KRSR), and protease-labile domains (e.g., APGL, VRN, or indeed oligoalanines that are degraded by elastase).
  • cell-adhesive peptides e.g., RGD, YIGSR, KQAGDV, KHIFSDDSSE, KRSR
  • protease-labile domains e.g., APGL, VRN, or indeed oligoalanines that are degraded by elastase.
  • Stimuli-responsive materials enabling the behaviour of the cells that reside within them to be controlled are vital for the development of instructive tissue scaffolds for tissue engineering.
  • This example describes the preparation of conductive silk foam-based bone tissue scaffolds that enable the electrical stimulation of human mesenchymal stem cells to enhance their differentiation towards osteogenic outcomes.
  • Bone tissues are hierarchically structured composite materials composed of both soft and hard matter (i.e., cell-rich vascularized soft tissue, and collagen-/hydroxyapatite-rich hard tissue). Bone conditions and disorders that require surgical intervention motivate
  • Biopolymer-based tissue scaffolds represent a particularly interesting class of biomaterials because of the versatile materials morphologies accessible via aqueous processing, and a variety of both polysaccharides and proteins have been investigated for their application as bone tissue scaffolds.
  • Natural silk proteins and recombinant silk-inspired proteins are frequently used as base materials for both drug delivery devices and tissue scaffolds with encouraging results both in vitro and in preclinical studies.
  • Electromagnetic fields may be employed for the non-invasive stimulation of bone growth, or as invasive implantable biointerfaces such as cardiac pacemakers and neural electrodes.
  • Biointerfaces based on conductive polymers (CPs), such as derivatives of polyaniline, polypyrrole or polythiophene, are of interest for both long term applications as low impedance coatings for electrodes with biomimetic mechanical properties and potentially for short term applications as drug delivery devices or tissue scaffolds for tissue
  • Pro-regenerative CP -based tissue scaffolds have been developed for various tissues.
  • Electrical stimulation of C2C12 mouse myoblasts (a common model for muscle cells) in vitro results in increased contractile activity and maturation relative to non- stimulated controls, [8] and therefore, C2C12-adhesive polythiophene-based hydrogels with biomimetic mechanical properties represent promising muscle tissue scaffolds.
  • conductive protein-based materials have been prepared previously. [6g] Some examples include those based on individual components of the extracellular matrix (e.g. collagen), [13] and decellularized tissues containing a variety of extracellular matrix proteins. Additionally, functionalization of spider [14] and silkworm [15] silks with polypyrrole yields anti-static silk textiles, or novel stimuli-responsive actuators.
  • HMSCs human mesenchymal stem cells
  • Aqueous solutions of silk fibroin were prepared in accordance with the literature. [17] Briefly, silk cocoons of B. mori silkworms were degummed by boiling in an aqueous solution of Na 2 C0 3 (0.02 M) for 20 min, followed by rinsing thoroughly with distilled water. The extracted silk fibroin was then dissolved in aqueous LiBr (9.3 M) at 60 °C for 4 h, and thereafter dialyzed against ultrapure water using a Slide-a-Lyzer dialysis cassette (MWCO 3,500, Life Technologies, Carlsbad, CA, USA) for 2 days. The solution was centrifuged at 9000 rpm (ca.
  • the petri dish was covered and left at room temperature for 48 h, after which the petri dish was immersed in water (2L) and the NaCl extracted for 2 days changing the water at least three times a day (minimum 6 washes). Samples were cut to lengths appropriate for the various subsequent experiments using a disposable biopsy punch and a razor blade.
  • the porous silk scaffolds were either stored in ultrapure water at 4 °C or lyophilized and stored at room temperature.
  • Interpenetrating networks of conductive polymers and silk fibroin were prepared by adaptation of the literature. [10d ' 18] Briefly, pyrrole was purified by passage over basic alumina. 2-hydroxy-5-sulfonic aniline (0.473 g, 2.5 mmol) was dissolved in HC1 solution (1 M, 50 mL) in disposable 50 mL centrifuge tubes. Pyrrole (0.175 mL, 2.5 mmol) was added and the sample cooled to 0 °C. 60 silk foams (4 mm in height and diameter) were added and incubated at 0 °C for 1 hour.
  • the porosity of the samples was measured by liquid displacement.
  • Hexane was used as the liquid as it does not swell or shrink the sample.
  • the sample was immersed in a known volume (V ⁇ ) of hexane in a graduated cylinder for 5 min.
  • the total volume of hexane and the hexane-impregnated sample was recorded as Vi-
  • the hexane-impregnated sample was then removed from the cylinder and the residual hexane volume was recorded as 3.
  • the total volume J 7 of the sample was:
  • V2-V1 is the volume of the polymer scaffold.
  • V ⁇ -V ⁇ is the volume of hexane within the scaffold.
  • the porosity of the scaffold ( ⁇ , %) was calculated from:
  • the swelling ratio (SR) and equilibrium water content (EWC) of the scaffold was calculated from:
  • Samples were mounted on a Scanning Electron Microscopy (SEM) stub and sputter coated with Pt/Pd (15 nm) using a Cressington 208 Benchtop Sputter Coater. All samples were imaged using a Zeiss Supra 40 VP field emission scanning electron microscope.
  • SEM Scanning Electron Microscopy
  • XPS X-ray photoelectron spectroscopy
  • XPS X-ray photoelectron spectrometer
  • a Thermo Scientific Nicolet 380 FTIR Spectrometer (Thermo Fisher Scientific Inc., USA) was used to record spectra in attenuated total reflectance (ATR) mode at 21 °C with a 1 cm -1 resolution and 128 scans (corrected for background and atmosphere using the software provided with the spectrometer). Samples were secured in position on the ATR crystal using the built-in clamp.
  • HMSCs were isolated from bone marrow aspirate (Lonza, Walkersville, MD) as described previously. [28] Briefly, aspirate from a male donor under 25 years old was combined with HMSC proliferation medium (MEM alpha with 10% fetal bovine serum (FBS), 1% antibiotic/antimycotic, 1% nonessential amino acids (NEAA)) and cultured at 37 C with 5% C0 2 in a humidified environment. Flasks were rocked every day to allow HMSCs to adhere and medium was added every 3-4 days until HMSCs reached 80% confluence. Non-adherent cells were removed via PBS washes and the HMSCs were cultured in proliferation medium until either passaged or frozen.
  • MEM alpha fetal bovine serum
  • NEAA nonessential amino acids
  • HMSC viability was assessed using a Cell Titer-Glo® luminescent cell viability assay kit (Promega, USA) in accordance with the with the manufacturer's protocol, using a Synergy HT Multi-Mode Microplate Reader (Biotek, USA) to analyze the luminescence of the samples.
  • a Cell Titer-Glo® luminescent cell viability assay kit Promega, USA
  • Synergy HT Multi-Mode Microplate Reader Biotek, USA
  • HMSC growth medium that was composed of: high glucose Dulbecco's Modified Eagle Medium (DMEM, 440 mL); fetal bovine serum (50 mL); antibiotic-antimycotic (5 mL); non-essential amino acids (5 mL), and 2 ng mL "1 basic fibroblast growth factor.
  • DMEM high glucose Dulbecco's Modified Eagle Medium
  • fetal bovine serum 50 mL
  • antibiotic-antimycotic 5 mL
  • non-essential amino acids 5 mL
  • 2 ng mL "1 basic fibroblast growth factor 2 ng mL "1 basic fibroblast growth factor
  • HMSCs were seeded at ca. 0.5 x 10 6 cells per foam, and incubated at 37 °C, 95 % humidity, and a C0 2 content of 5 %.
  • osteogenic medium that was composed of: high glucose Dulbecco's Modified Eagle Medium (DMEM, 425 mL); fetal bovine serum (50 mL); antibiotic-antimycotic (5 mL); non-essential amino acids (5 mL), dexamethasone (100 nM), ⁇ -glycerol phosphate (10 mM) and ascorbic acid (50 ⁇ ). Thereafter the osteogenic medium was aspirated and replaced every 2 days until the samples were analyzed.
  • DMEM high glucose Dulbecco's Modified Eagle Medium
  • fetal bovine serum 50 mL
  • antibiotic-antimycotic 5 mL
  • non-essential amino acids 5 mL
  • dexamethasone 100 nM
  • ⁇ -glycerol phosphate 10 mM
  • ascorbic acid 50 ⁇
  • HMSC growth medium that was composed of: high glucose Dulbecco's Modified Eagle Medium (DMEM, 440 mL); fetal bovine serum (50 mL); antibiotic-antimycotic (5 mL); non-essential amino acids (5 mL), and 2 ng mL "1 basic fibroblast growth factor.
  • DMEM high glucose Dulbecco's Modified Eagle Medium
  • fetal bovine serum 50 mL
  • antibiotic-antimycotic 5 mL
  • non-essential amino acids 5 mL
  • 2 ng mL "1 basic fibroblast growth factor 2 ng mL "1 basic fibroblast growth factor
  • HMSCs were seeded at ca. 0.5 x 10 6 cells per foam, and incubated at 37 °C, 95 % humidity, and a C0 2 content of 5 %.
  • osteogenic medium that was composed of: high glucose Dulbecco's Modified Eagle Medium (DMEM, 425 mL); fetal bovine serum (50 mL); antibiotic-antimycotic (5 mL); non-essential amino acids (5 mL), dexamethasone (100 nM), ⁇ -glycerol phosphate (10 mM) and ascorbic acid (50 ⁇ ).
  • DMEM high glucose Dulbecco's Modified Eagle Medium
  • fetal bovine serum 50 mL
  • antibiotic-antimycotic 5 mL
  • non-essential amino acids 5 mL
  • dexamethasone 100 nM
  • ⁇ -glycerol phosphate 10 mM
  • ascorbic acid 50 ⁇
  • the tips of the copper wires attached to the foams were wound around alligator clip- terminated wires attached to the multipotentiostat (CH Instruments, Austin, TX, USA).
  • the counter and reference electrodes were connected together and clipped to the copper wire protruding from one end of the sample, and the working electrode was clipped to copper wire protruding from the other side of the sample.
  • Wires and alligator clips were secured in position with adhesive copper tape (Ted Pella, Inc., Reading, CA, USA) and wrapped in Parafilm® to render them electrically insulating and waterproof (i.e. suitable for use inside an incubator).
  • a potential step of 100 mV/mm was placed across the samples for the duration of 4 h per day for 6 days, after which the wires were disconnected and the substrates cultured as normal. Throughout the electrical stimulation experiments the osteogenic medium was aspirated and replaced every 2 days. Thereafter the osteogenic medium was aspirated and replaced every 2 days until the samples were analyzed.
  • the DNA content and Alkaline Phosphatase (ALP) activity of samples that were broken up in a buffer of 0.2% Triton X-100 were quantified concurrently, using the PicoGreen® assay (Life Technologies, Thermo Fisher Scientific Inc., USA) for DNA quantitation in accordance with the manufacturer's protocol, a SensoLyte® >NPP Alkaline Phosphatase Assay Kit (AnaSpec, Inc., Freemont, CA, USA) for ALP quantitation in accordance with the manufacturer's protocol, and a Synergy HT Multi-Mode Microplate Reader (Bio-tek US, Winooski, VT).
  • Hematoxylin & Eosin (H&E) staining samples were dehydrated in xylene and ethanol, rehydrated in water, dipped in Harris Hematoxylin solution to stain nuclei, rinsed in water and placed in Scott's solution until they turned blue, rinsed again in water, dipped in Eosin solution to stain the ECM, then dehydrated prior to being covered with a coverslip in xylene-based mounting media.
  • Alizarin red staining samples were dipped in 1% Alizarin red stain with a pH of 4.1-4.3 for 2 minutes, followed by 20 dips in acetone, followed by 20 dips in 50/50 Acetone/Xylene, then
  • Histomount was used to cover each slide and the samples were left to dry overnight before imaging (images are representative of 3 samples).
  • the interpenetrating network of a self-doped CP within the silk foam matrix was composed of pyrrole and 2-hydroxy-5-sulfonic aniline (Figure 1B), [20] and their polymerization within the silk foams was initiated by ammonium persulfate and ferric chloride. [21] When the scaffolds were homogeneously coloured, they were washed thoroughly with water and ethanol to remove the by-products that were not within or attached to the silk matrix (e.g. initiators, monomers, oligomers and polymers).
  • the resulting conductive foams had the same pore size distributions, swell ratio and equilibrium water content as non- conductive foams ( Figure IB, Table 1), however, the porosity of the foams (as determined by hexane displacement) was moderately reduced because of the presence of an interpenetrating network of the CPs within the hydrogel-like matrix of inter-/intra-molecularly crosslinked silk proteins that constitute the foam (Table 1).
  • Bone tissue engineering is a vibrant field of research, and as noted above, an enormous variety of materials have been investigated for bone tissue engineering, and silk proteins are a class of materials that has shown great promise both in vitro and in vivo in preclinical trials.
  • silk proteins are a class of materials that has shown great promise both in vitro and in vivo in preclinical trials.
  • electrical stimulation increased quantities of calcium and collagen deposited in the scaffolds, which is an important step towards the formation of calcified extracellular matrix associated with bone.
  • ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
  • a concentration range of "about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range.
  • the term “about” can include traditional rounding according to significant figures of the numerical value.
  • the phrase "about 'x' to y includes “about 'x' to about 'y” ⁇

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Abstract

Des modes de réalisation de la présente invention concernent des mousses conductrices (par exemple, des mousses de soie, des mousses de polymère), des procédés de fabrication de la mousse conductrice, un procédé d'utilisation de la mousse conductrice, et des éléments similaires.
PCT/US2016/041889 2015-07-15 2016-07-12 Échafaudages électroactifs et procédés d'utilisation d'échafaudages électroactifs Ceased WO2017011452A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109180995A (zh) * 2018-09-26 2019-01-11 德清舒华泡沫座椅有限公司 一种导电改性海绵
WO2019071033A1 (fr) * 2017-10-05 2019-04-11 The Board Of Trustees Of The Leland Stanford Junior University Échafaudage composite de nanofibres de graphène/carbone conductrices, son utilisation pour ingénierie tissulaire neuronale et son procédé de préparation
WO2024186903A1 (fr) * 2023-03-06 2024-09-12 Outer Biosciences, Inc. Systèmes et processus de croissance et d'analyse de tissu cutané

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5431998A (en) * 1993-05-14 1995-07-11 Lockheed Corporation Dimensionally graded conductive foam
US6214260B1 (en) * 1997-11-10 2001-04-10 World Properties, Inc. Conductive elastomeric foams by in-situ vapor phase polymerization of pyrroles
US20020034796A1 (en) * 1998-09-18 2002-03-21 Venkatram Shastri Electroactive materials for stimulation of biological activity of stem cells
US20080038236A1 (en) * 2006-03-06 2008-02-14 Artecel Sciences, Inc. Biocompatible scaffolds and adipose-derived stem cells
US20090171406A1 (en) * 2007-12-12 2009-07-02 Cardiac Pacemakers, Inc. Electrically conducting scaffolds for cell-based pacing
US20140094410A1 (en) * 2003-01-07 2014-04-03 David L. Kaplan Silk fibroin materials and use thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5431998A (en) * 1993-05-14 1995-07-11 Lockheed Corporation Dimensionally graded conductive foam
US6214260B1 (en) * 1997-11-10 2001-04-10 World Properties, Inc. Conductive elastomeric foams by in-situ vapor phase polymerization of pyrroles
US20020034796A1 (en) * 1998-09-18 2002-03-21 Venkatram Shastri Electroactive materials for stimulation of biological activity of stem cells
US20140094410A1 (en) * 2003-01-07 2014-04-03 David L. Kaplan Silk fibroin materials and use thereof
US20080038236A1 (en) * 2006-03-06 2008-02-14 Artecel Sciences, Inc. Biocompatible scaffolds and adipose-derived stem cells
US20090171406A1 (en) * 2007-12-12 2009-07-02 Cardiac Pacemakers, Inc. Electrically conducting scaffolds for cell-based pacing

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LI ET AL.: "Efficient Synthesis of Intrinsically Conducting Polypyrrole Nanoparticles Containing Hydroxy Sulfoaniline as Key Self-Stabilized Units", J. PHYS. CHEM. C, vol. 113, 2009, pages 21586 - 21595, XP055345009 *
MCCULLEN ET AL.: "Application of Low-Frequency Alternating Current Electric Fields Via Interdigitated Electrodes: Effects on Cellular Viability, Cytoplasmic Calcium, and Osteogenic Differentiation of Human Adipose-Derived Stem Cells", TISSUE ENGINEERING PART C: METHODS., vol. 16, no. 6, November 2010 (2010-11-01), pages 1377 - 1386, XP055345011 *
PELTO ET AL.: "Novel Polypyrrole-Coated Polylactide Scaffolds Enhance Adipose Stem Cell Proliferation and Early Osteogenic Differentiation", TISSUE ENGINEERING PART A., vol. 19, no. 7 -8, March 2013 (2013-03-01), pages 882 - 892, XP055345006 *
ROCKWOOD ET AL.: "Ingrowth of Human Mesenchymal Stem Cells into Porous Silk Particle Reinforced Silk Composite Scaffolds: An In Vitro Study", ACTA BIOMATER., vol. 7, no. 1, January 2011 (2011-01-01), pages 144 - 151, XP027448756 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019071033A1 (fr) * 2017-10-05 2019-04-11 The Board Of Trustees Of The Leland Stanford Junior University Échafaudage composite de nanofibres de graphène/carbone conductrices, son utilisation pour ingénierie tissulaire neuronale et son procédé de préparation
US12285544B2 (en) 2017-10-05 2025-04-29 The Board Of Trustees Of The Leland Stanford Junior University Conductive graphene/carbon nanofiber composite scaffold, its use for neural tissue engineering and a method of preparation thereof
CN109180995A (zh) * 2018-09-26 2019-01-11 德清舒华泡沫座椅有限公司 一种导电改性海绵
WO2024186903A1 (fr) * 2023-03-06 2024-09-12 Outer Biosciences, Inc. Systèmes et processus de croissance et d'analyse de tissu cutané

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