WO2014153610A1 - Surfaces de liaison de facteur de croissance et utilisations de celles-ci - Google Patents

Surfaces de liaison de facteur de croissance et utilisations de celles-ci Download PDF

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WO2014153610A1
WO2014153610A1 PCT/AU2014/000326 AU2014000326W WO2014153610A1 WO 2014153610 A1 WO2014153610 A1 WO 2014153610A1 AU 2014000326 W AU2014000326 W AU 2014000326W WO 2014153610 A1 WO2014153610 A1 WO 2014153610A1
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cells
growth factor
amine
glycosaminoglycan
substrate
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PCT/AU2014/000326
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David Robinson
Robert D. SHORT
Jason Whittle
Louise Smith
David Steele
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University Of South Australia
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Publication of WO2014153610A1 publication Critical patent/WO2014153610A1/fr

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    • 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
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N5/0656—Adult fibroblasts
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • 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
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
    • C12N2502/094—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells keratinocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides

Definitions

  • the present invention relates to substrates having growth factors bound to a surface thereof, and to uses of those substrates.
  • GFs Growth factors
  • GFs are regulators of cell growth both in vitro arid in vivo and they have a wide range of biological functions which affect cell proliferation, .migration, angiogeriesis, differentiation and apoptosis. GFs are secreted by cells and function by binding to cell surface receptors.
  • GFs are important for regulating a variety of cellular processes.
  • GFs pla an important role in wound healing which is a complex multicellular process that involves fibroblasts, keratinocyles and endothelial cells.
  • Normal wound healing is regulated by a number of GFs, cytokines and ehemokmes.
  • FGF-2 fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • KGF keratinocyte growth factor
  • EEF epidermal growth factor
  • GFs have an effect on human embryonic stem cell (hES) differentiation.
  • Schulinder et al investigated the effects of eight different growth factors,, including FGF-2 and EG! 7 , on hES. cells and showed that different growth factors produced different cell lineages. The GF effects fell into three main groups; those that produce mesodermal cells, ectodermal cells or endodermal cells (Schuldiner et a!. 2000).
  • Age-reiated macular degeneration is another area where GFs may be used. AMD results from damage to the retinal pigment epithelium (EPF.) whereby the central vision is gradually lost due to pigmentary abnormalities and accumulation of waste materials (drusen), resulting in RPE detachment, leakage and choroidal neovascularisation (Fine el al. 2000).
  • EPF. retinal pigment epithelium
  • drusen waste materials
  • restoration of vision could utilise the transplantation of a cell sheet of healthy, functional RPE.
  • Such a procedure requires the in vitro culture of a small biopsy of healthy tissue in an environment conducive to cell growth and proliferation and GFs may be utilised in the media to aid in cell proliferation.
  • a method of immobilising a growth, factor on a substrate comprising:
  • a substrate having a growth factor immobilised thereon comprising an amine functionalised surface on the substrate, at least one glycosaminoglycan bound nem-covalentiy to the amine functionaiised surface, and at least one growth factor bound to the glycosammoglyean,
  • a method of growing cells on a substrate comprising:
  • glycosaminoglycancorrectalised surface with a medium, comprising one or more growth factors and the cells to be cultured under conditions suitable for growing the cells.
  • a method of growing cells on a substrate comprising:
  • the amine fiiiictionalised surface is formed by plasma polymerisation of a primary amine.
  • the primary amine may he ailylamine.
  • the growth factor is a fibroblast growth factor, such as FGF-2.
  • the growth factor is platelet derived growth factor (PDGF).
  • the giyoosamiiiogiycan is selected from the group consistmg of heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate, keratin sulfate, and hyaluronic acid.
  • the glycosammoglycaa may also be a fragment or derivative of any of the aforementioned glycosaniirraglycans.
  • the glycosaminoglycan is heparan sulfate, hi other specific embodiments, the glycosaminoglycan is heparin.
  • the cells are epithelial cells. In specific embodiments, the epithelial cells are retinal pigment epithelium (RPE) cells.
  • the cells are fibroblast cells.
  • the fibroblast cells are dermal fibroblast cells.
  • a tissue engineered scaffold comprising:
  • the method further comprises incubating the scaffold having the growth factor bound to the surface with cells of interest under conditions to proliferate cells an the surface.
  • a tissue engineered scaffold having a growth factor immobilised thereon, the scaffold comprising an amine ftmetionalised surface thereon, at least one glycosaminoglycan non-covalentl bound to the amine funetionalised surface, and at least one growth factor bound to the glycosaminoglycan.
  • the glycosaminoglycan is selected from the group consisting of heparin, heparan sulfate, derraatan sulfate, choadroitin sulfate, keratin sulfate, and hyaluronic acid.
  • the glycosaminoglycan may also be a fragment or deri vative of any of the
  • glycosaminoglycans aforementioned glycosaminoglycans.
  • the glycosamin glyean is heparin.
  • the tissue engineered scaffold is a skin scaffold.
  • the growth factor is a fibroblast growth factor and the cells are dermal fibroblast cells and/or keratinocyte cells.
  • Figure 1 shows XPS spectra (surve and narrow scan) of ppAA coated onto the 48 well plates.
  • the surface contains carbon (C 76.58%), nitrogen (N 15.69%) and oxygen (Q 7.73%) which gives an N/C ratio of 0.2O
  • GAGs HS 20(tyg/mi, DS 50ug/ml, CS 800pg/nil
  • Figure 4 shows a plot of ARPE-19 cell viability on plasma polymer coated multiwell plate in serum-reduced medium supplemented with heparan sulfate (200 ⁇
  • Cells were cultured in medium not supplemented with GAGs as a control ( ⁇ SE , n ⁇ 4).
  • Figure 5 shows plots of ARPE-19 cell viability on plasma polymer coated multiwell plate in serum-reduced medium.
  • Figure 7 shows photomicrographs of the expression of " RPE65 (A-D) and the phagocytosis of latex beads (E-H) by ARPE-l 9 at 5 days culture on plasma polymer coated glass coverslips alone (A and E), pre-incubated with all three GAGs together (B and F), with PDGF-CC (5000 ng/ml) immobilised on the surface via HS (C and G) and with FGF-2 (50 ng/riil) supplemented medium (D and H).
  • Figure 12 shows plots of HFB and HFM compared at 9 days culture at all serum containing percentages to analyse the differences in cell numbers when biomolecules are bound compared to adde i to the media ( ⁇ SEM, n ⁇ 3).
  • Figure 13 shows (a) schematic of the plasma reactor; (b) method of mounting scaffolds for coating; and (c) scaffold preparation for TOF-5IMS analysis.
  • Figure 15 shows negative ToF-SMS images taken using the unbundled mode, Total negative ion images and images for the total intensity of [C0 2 B " (m z 45).
  • CaHeOj " (m z 113) and Cr,RnQ (m/z 131)], [CK (m z 26) and CNO- ' (m/z 42)] and SQ 2 ⁇ (m z 64) and S0 4 (m/z 96)] were acquired from (!) PCL, (2) PCL/ppAA and (3) PCL/ppAA'Heparin cross sections.
  • Scale bar 100 microns.
  • Figure 16 shows high resolution negative ToF-SIMS spectra recorded in the region around 32 m/z where S " (3 i .97 amii) and G? (31.99 amn) can be observed, (a) PCL as prepared, (b PCL/ppAA and (c) PCL/ppAA/Heparin scaffolds.
  • Figure 18 shows a resazurin assay after liir HDFs incubation (mean ⁇ 1 SD, a-3).
  • Figu e 20 shows the formation of dermal and epidermal layers after 5 weeks culture of primar HDFs and primary keratinocytes on a 3D scaffold, for conditions 1 : PCL, 2: PCL+ ppAA and 3 :
  • PCL+ppAA+ GAG+GF The grid is set Out to show both H&B staining and specific antibody staining for paa-cytokeratin with the nuclear stain DAPI a a counter stain. Scale bars 100 microns.
  • Figure 21 shows XPS spectra of washed and unwashed allylamine surfaces produced under condiiioas for plasma coating.
  • Figure 22 shows representative negative ion ToF-SIMS spectra collected from regions of interest (ROIs) on the surface of (a) PCL, (b) PCL/ppAA and (c) PCL ppAA Heparin scaffolds. Characteristic peaks assignments for PCL are shown in blue (#), for PCL/ppAA are shown in red ( ⁇ ) and for
  • Figure 23 is a plot showing the number of HDFs cells attached to scaffolds of conditions 1 , 2 and 3 after Alix10 3 seeded.
  • Figure 24 shows a photomicrograph showing fibroblasts grown into an allylamine scaffold for 4 weeks and stained with both DAPI and pan-cytokeraiin as a control.
  • a method of irmuobilising a growth factor on a substrate comprises forming an amine functionalised surface on the substrate.
  • the amine functionalised surface is then contacted with a glycosaminoglycan under conditions to non-covalent!y bind the glycosaminoglycan and form a glycosaminoglycan functionalised surface.
  • the glycosaminoglycan functionalised surface is then contact with a growth factor under conditions to bind the growth factor to the surface.
  • Glycosaminoglycans such as heparan sulfate, which is a structural mimetic of heparin, have been shown to bind PGP-2, VBGF and other important growth factors (Raman et al 2005),
  • Glycosaminoglycans have also been shown to enhance growth factor interactions and have also been shown to protect them from degradation in the EC (Raman et at, 2005, Zem et al. 2010). They also exhibit cytokine and chemokine binding, which are important in cell migration, inflammation and immune response, and in the binding to leukocytes and endothelial cells in areas of inflammation.
  • the glycosaminoglycan dermatan sulfate is the most abundant giycosaminoglycan in (he skin and has been shown to bind KGF, increasing keratinocyte production in the wound environment (Trowbridge et al 2002).
  • Glycosaminoglycans also bind fibronectirt, vitronectin and laminin which may be important in aiding cell attachment to scaffolds or other cell devices (Hilenian et. al. 1 98, Sun et al. 2005),
  • a modified surface consisting of giycosaminqglycans that can bind growth factors, cytokines, chemokines and other important biomoteeules could be a useful tool (Zerti et al. 2010).
  • Surfaces may be made with localised modifications with different growth factors to induce cell proliferation and differentiation in a spatially controlled fashion. This approach could be applied not only in the culture of patient cells, but also in areas of cancer research where the role of growth factors on cancer progression is an important research area and the growth factor expense is high.
  • the response of cancer cells to growth factors is greater, as a result of an increased proliferative response as the need for specific growth factors to induce specific responses are lost (Goustin et al. 1986, Chen et al. 2010).
  • Research is ongoing into cancer cells and the changes that occur to the interaction between growth factors and their receptors and how glycosaminoglycans influence tumour progression (Yip- at al. 200,6).
  • the giycosaminoglycan is non-eova!ently bound: to the amine funcfionalised surface.
  • a nen-eovalent interaction differs from a. covalent bond in that it does not involve the sharing of electrons. Non-covalent interactions may res'ult from electrostatic, -effects, van der Waals forces, and/or hydrophobic effects.
  • the present invention is therefore distinguished irohi prior art methods in which glycosaminoglycans are covalently bound to surfaces. For example, Yoon et.
  • PLGA poly(B,LTactide-cc- glycolide)
  • the method involves covalently banding a PEG-bis-amine to free catboxyl groups on the surface of the PLGA scaffold usin EDC/NHS crosslinking to covalently bond the PEG- bis-amine to the surface via amide bonds.
  • Heparin was then covalently bonded to the PEG-amine functionaiised surface using coupling agents, The modified scaffold was then used for the sustained release of basic fibroblast growth factor over a period of 20 days.
  • Wissihk et al Wissihk et al.
  • Wissink et al. covalently bond heparin to cross-linked collagen films by activating free carboxyl groups using EDC and NHS and then reacting them with free amino groups on the collagen film to covalently bond the heparin to the surface via amide bonds
  • Liu ei al. describe the formation of a conjugated hyaluronate-heparin gel (Liu el al., 2002).
  • Hyahironate is activated by oxidation and primary amine groups were introduced to the HA by reductive animation with NaB3 ⁇ 4CN in the presence of ethylenediarnine. Subsequently, heparin was covalently bonded, to the HA by reductive animation of .heparin in the presence of the amine activated HA. Similar methodology is also described in international patent application WO 2000/078356 Al (Qrquest Inc). Whilst these prior art methods can be used to immobilise heparin on the surface of a material, the- immobilisation processes are complicated and require the use of coupling agents to covalently bond the heparin to ; the surface.
  • the substrate that is coated using the methods described herein may be any suitable material such as polystyrene, polyethylene, polypropylene, metals, alloys, ceramics, and the like.
  • the substrate may be an FXISA plate, multi-well plate, membrane, sheet, rod, strip, plate, slide, bead, pellet, disk, tube, sphere, chip, plate and dish, or any other suitable shape.
  • the substrate may be an implant.
  • the substrate may be a complex three dimensional shape.
  • the amine functionortaiised surface is conveniently formed .by plasma polymerisation of a primary amine.
  • plasma polymer films of ally) amine (ppAA) deposited onto microtitre plates can be used to non-covaieiitly bind glycosaminoglycans, through positive charges derived from (he primary amine groups present in the allylamine monomer (Marson ei al, 2009).
  • Plasma polymerisation is generally achieved under reduced pressure, with the basic requirements being a vessel containing the vapour of an organic compound at low pressure and some means of coupling energ into the system, (typically radio frequency radiation at 13.56MHz).
  • plasma polymers do not contain regular repeat units and there is a degree of fragmentation of the monomer precursor during deposition, which leads to a relative loss of functionality. This depends on the processing conditions used. Under the conditions of low plasma power highly functionalised surfaces may be produced (Gengenbach et al. 1996, Calderon et al 1998, Aizawa ei al. 2000, Whittle et al, 2000, Zhang et al, 2003, Zhang et al. 2005).
  • the primary amine may be any amine thai is sufficiently volatile to form a vapour in the plasma chamber.
  • the amine groups formed on the surface of the substrate are able to non-cQvalently bind glycosammoglycaris through positive charges derived fro ' ttj the primary amine group.
  • Suitable primary amines include allylarnine, propylamine, methylamine, ethylamme, heptylamine, butylamkie, or any other primary aliphatic or aromatic amine which may include one or more double bonds (e.g. allylarnine), may be cyclic (e.g. cyclopentylamine, aniline), may be a diamine (e.g. diam noprepane, ethylenediamme).
  • the glyeosammoglycan may be any long unbranched polysaccharide consistin of a repeating disaecharide unit that is capable of binding amine groups and gro wth factors.
  • the glyeosammoglycan may be selected from the group consisting of heparin, heparan sulfate, dermatafi sulfate, chondroitin sulfate, keratin sulfate, and hyaluronic acid.
  • the glycosaminoglycan may also be a fragmeut or derivative of any of the aforementioned giycosaminoglycans wliich retains its ability to biisd the growth factor.
  • the glycosaminoglycan may a 10-mer ⁇ 10 disaccharides) of one of the aforementioned giycosaminoglycans that retains its ability to bind growth factors.
  • the amine functionalised surface is contacted with the glycosaminoglycan under conditions to bind the glycosaminoglycan. Specifically, a solution of the glycosaminoglycan is incubated with the substrate comprising the amine functionalised surface for a period of 1 to 24 hours.
  • the growth factor (OF) ma be a vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF) or hepatocyte growth f actor (HGF).
  • VEGF vascular endothelial growth factor
  • PDGF platelet derived growth factor
  • FGF fibroblast growth factor
  • TGF transforming growth factor
  • HGF hepatocyte growth f actor
  • glycosaminoglycan functionalised surface may be protected from degradation and, therefore, have an extended half -life compared to growth factor in media, thereby reducing costs of cell culture (Francli et al 2001)
  • the growth factor is FGF-2
  • FGF-2 is a growth factor that promotes proliferation of fibroblast cells in the dermis.
  • FGF-2 is bound to the glycosaminoglycan functionalised surface by incubating a solution of FGF-2 in a medium such as PBS at 37*C, The incubation time may be from about 1 hour to about 4 hours. In embodiments, the incubation time is 2 hours.
  • the concentration of FGF-2 in the solution that is contacted with the glycosaminoglycan functionalised surface may be from about 1 ng/ml to about 2500 ng/ml, such as 10 ng/ml, 50 ng/ nl, 100 ng/ml, 1000 ng/ml or 2000 ng/ml.
  • ng/ml ng/ml
  • 50 ng/ nl 100 ng/ml
  • 1000 ng/ml or 2000 ng/ml For proliferation of Human Dermal Fibroblast (HDF) cells we found no significant difference between adding 10 or lOOng/m as a similar level of proliferation was observed.
  • the growth factor is PDGF-CC
  • concentration ofPDGF-CC in the solution that is contacted with the glycosaminoglycan functionalised surface may be from about 1 ng/ml to about 5000 ng/ml, such as 125 ng ml or 5000 ng/ml.
  • TE3 ⁇ 4e Substrate may be used in maintaining or proliferatirig cells.
  • the cells are epithelial cells.
  • the epithelial cells are retinal pigment epithelium (RPE) cells.
  • the cells are fibroblast cells.
  • the fibroblast cells are dermal fibroblast cells.
  • keratinoeyfes embryonic stem cells (ES), mesenchymal stem cells (mES), induced pluripotent cells (IPS), endothelial progenitor cells (EPCs), regulatory T cells (T-regs), islet cells, and other human primary cells.
  • ES embryonic stem cells
  • mES mesenchymal stem cells
  • IPS induced pluripotent cells
  • EPCs endothelial progenitor cells
  • T-regs regulatory T cells
  • islet cells and other human primary cells.
  • Also disclosed herein is a method of growing cells on a substrate, the method comprising:
  • glycosaminoglycan functionalised surface with a medium comprising one or more growth factors and the cells to be cultured under conditions suitable for growing the cells.
  • the cells are retinal pigment epithelium (RPE) cells
  • the glycosaminoglycan is heparan sulphate and the growth factor is FGF-2.
  • the conditions suitable from growing cells may be > 100 hours, such as about 120 hours.
  • a method of growing cells on a substrate comprising:
  • the cells are human dermal fibroblast (BDF) cells, the glycosaminoglycan is heparin and the growth factor is FGF-2.
  • the conditions for growing cells may include the use of suitable serum.
  • the serum concentration is ⁇ 5%, such as 2.5% or 1 ,25%,
  • Modified surfaces can also be transferred to 3D synthetic scaffolds to aid the production of models containing both dermis and epidermis. These could be used to investigate non-healing wounds in a lab environment without the need for animal models and to eliminate the rate limiting steps of gathering skin from patients in current human skin equivalent models. Additionally, for patients suffering the advanced form of AMD, restoration of vision could utilise the transplantation of a ceil sheet of healthy, functional PE. Such a procedure requires the in vitro culture of a small biopsy of healthy tissue in an environment conducive to cell growth and proliferation. The ultimate goal of this work being a more complete and more ethical skin model for lab based experiments on non-healing wounds (Sun et al. 2005, Commandeur et al. 2012, Moliararnzadeh et at 2012).
  • Tissue engineered (TE) scaffolds have become an important area of bioengirieermg to help deal with ageing populations and the unmet need ibr organs for transplantation.
  • Many scaffolds have been designed that mimic the extracellular matrix in order to encourage growth of harvested ceils from a range of tissue types such as skifti bone and cartilage, Therefore the provision of tissue engineered products is a rapidly growing field that has the potential to improve patient outcomes. For example, improved scaffolds are needed to treat terns and chrome wounds.
  • a common feature is the production in vitro of a cell --seeded polymer construct, prior to implantation. Production is costly arid time consuming.
  • tissue engineered scaffolds for the treatment of organ failure or injury is currentl a niaj or area of interest.
  • a particular aim of this field is to provide organs grown from implantable scaffolds using the patient's own cells, thereby eliminating the risk of immunological rejection (Yang et al., 2002). It is necessary for the cells to be able to migrate, proliferate and differentiate into the scaffolds and therefore understanding of the required scaffold properties is highly important in allowing the cells to produce the desired tissue or organ (Place et al., 2009),
  • a range of different scaffolds and cell types to achieve tissue engineering goals for bone, muscle, heart, cartilage and skin tissues have been explored in vivo and in vitro.
  • One of the most successful areas of research has been in regenerative skin products and treatments.
  • Tissue engineered products for the treatment of wounds and aid healing in patients suffering from diabetic foot ulcers which are non-healing are available commercially (Waiiiwright et al., 1995; Eaglstein and Falange, 1997; Gentzkow et al., 1996).
  • Dcmi graftTM is a scaffold containing cultured fibroblast cells while ApligraF M contains both fibroblasts and keratiiiocytes. Both, of these products are produced by expandin cells in Culture and then seeding onto Scaffolds which are used; for treatment. The fabrication of the seeded scaffolds is therefore a time ' consuming process.
  • fibroblasts In a typical healing wound, human dermal fibroblasts (HDFs) secrete growth factors (GFs) and other essential biomoleeules known to support keratinocyte migration and formation of an epidermal layer. Additionally, fibroblasts play an important role in the maintenance of the basement membrane between the dermal and epithelial layers.
  • the basement membrane is largely composed of collagen VII fibrils which anchor the cells together, as well as other major proteins including laminin ⁇ , ⁇ 3, ⁇ 2 and the heparan sulphate proteoglycan, perlecan.
  • the GAG heparan sulphate is an important biomolecule in extra cellular matrix (ECM) and in wound healing, due to its ability to bind and protect GFs, including fibroblast growth factor (FGF)3 ⁇ 4 which have a short half-life.
  • ECM extra cellular matrix
  • FGF fibroblast growth factor
  • tissue engineered products particularly in the context of engineering for the skin
  • substrates comprise typically, polystyrene, caprolactone, glycolide or lactide based scafiolds which are engineered to mimic ECM architecture (Peppas and Langer, 1994; Famigia et ai., 2012; Milbura et at., 2008; Powell et a/., 2008).
  • Production is sometimes aided by the addition of biomoleeules to synthetic scaffolds, or by the replacement of synthetic substrate materials with naturally occurring macromolecules such as type 1 collagen, GAGs or chitosao ( ohn and Laiiger, 1987; l ' )»;m at.. 2007; Quirk et at, 2Q01).
  • a method of forming a tissue engineered scaffold comprises forming an amine functionalised surface on the scaffold by plasma polymerising a primary amine monomer on the surface.
  • the primary amine is allylamine
  • the amine functionalised surface on the scaffold is then contacted with a glycosaminoglycan Under conditions to non-covakm y bind the glycosaminoglycan to the amine functionalised suriace and form a glycosaminoglycan functionalised surface, hi embodiments, the glycosaminoglycan is heparin.
  • the glycosaminoglycan functionalised surface is then contacted with a growth factor under conditions to bind the growth factor to the surface.
  • the growth factor is fibroblast growth factor (FGP-2).
  • the method further comprises incubating the scaffold having the growth factor bound to the surface with cells of interest under conditions to proliferate cells on the surface.
  • Cells of interest inclnde fibroblasts and keratinocytes.
  • a tissue engineered scaffold having growth factor immobilised thereon, the scaffold comprising an amine functionalised surface thereon, at least one glycosaminoglycan non-covalenfly bound to the amine functionalised surface, and at least one growth factor bound to the glycosaminoglycan.
  • Example 1 Preparation of substrates having amine functionalised surfaces
  • Substrates having amine ftmetionalised surfaces were prepared using a cylindrical reactor chamber (LewVac, Burgess Hill, UK) with an approximate volume of 27 litres. RF power was supplied via an impedance matching unit to an internally located driven electrode. Substrates were puinped down to a base pressure of 4x10 "4 mbar using a rotary pump (BOC Edwards, Crawley, UK) and a liquid nitrogen cold trap (MDC Vacuum Products Corp., Hayward, CA) located between the pump and the reactor chamber.
  • a rotary pump BOC Edwards, Crawley, UK
  • AllyiaHiine vapour was introduced into the reactor via a needle valve at a flow rate of 5secm.
  • Plasma was ignited with art input power of 5 Watts (read from the RF power supply), and the samples were coated for 30 minutes.
  • the resultant plasma polymer surface had a thickness greater than 10 nm and the surface chemistry was analysed by XPS.
  • GAGs glycosaminogiycans
  • Heparan sulfate (HS) 200 ⁇ / ⁇
  • dermatan sulfate (DS) 50 pg ml
  • CS cliondroitin sulfate
  • ARPE-19 cells were maintained in complete medium: Dulbeeco's Modified Eagles
  • DMEM/F12 Ham's F12 Nutrient Mixture (1: 1) (Gibco, Invitrogen Australia) with 10% fetal bovine serum (FBS) (AusGene, Arundel, Australia), lOOIU/ml of penicillin and 100 ⁇ g/ml streptomycin (Gibco, Invitrogen Australia).
  • FBS fetal bovine serum
  • streptomycin 100 ⁇ g/ml streptomycin
  • FGF-2 a d recombinant human PDGF-CC were immobilised on this IIS for 2 hours at 37°C, Both high and low concentrations of protein solution for immoblHsalion were studied: for FGF-2; 2000 ng ml and 50 ng/ml and for PDGF-CC; 5000 ng/ml and 125 ng/ml. In a control sample neither HS nor protein were immobilised. Subsequently, unbound proteins were removed by washing with PBS before ARPE-19 cells were seeded at a density of .10,000 cells/well.
  • Cell viability was analysed following 24, 8 and 120 hours of culture by measurement of the concentration of the reduced form of fesazerin using the following- protocol, Cells were incubated for 4 hours in a 10% solution of resazurin stock (1 10 ⁇ / ⁇ 1.) in DMEM/FI2 (1 :1) medium and dye absorbance read at 570 and 600 nm using a microplate reader (FLUOstar OPTIMA manufactured by BMG LABTECH).
  • Cell morphology was determined by confocal observation of fluorophore labelled actin filaments and nuclei. Stained cells were observed using a Nikon AIR confocal laser microscope system with motori ed Eclipse T ' i inverted microscope. Prior to staining cells were fixed With 4%
  • Assay Kit (IgG PE) comprising of latex beads coated with phycoerythrin (PE)-labetiedrabbit-lgG (Cayman) was employed. Cells were cultured on the prepared surfaces for 4 days prior to a suspension of latex beads in medium (1: 100) being added and the cells cultured for one additional day. Subsequently, ceils were washed with PBS and stained with 3,3'-dipentyloxacarbocyaniiie iodide (DiOCj) (4 ⁇ /ml medium) for an hour in an incubator at 37°C. Finally, cells were washed twice in PBS, fixed in 4% paraformaldehyde for 10 minutes and mounted on a glass slide for examination.
  • IgG PE phycoerythrin
  • ARi'i- 19 papulations were examined after 120 hours of culture.
  • Cell morphology was determined via phallotoxin staining of F-aatin, Additional cell phenotype was examined by the ability of cell populations, stained green with DiOC 5 , to phagocyte phycoerytrin: (PB)-labelled latex beads and express of the visual cycle protein RPE65.
  • GAGs can be immobilised to an amine ftmctionalised plasma polymer surface via charge interactions between the positively charged modified surfaces and the negatively charged sulfate groups contained within the GAGs.
  • GAGs such HS, DS or CS can be incubated overnight with the modified plates and they are functionally retained as shown by ELIS A protocols with the known HS binding the FGF-2 and PDGF-CC proteins.
  • ELIS A protocols with the known HS binding the FGF-2 and PDGF-CC proteins.
  • EGF epidermal growth factor
  • DMEM Modified Eagles Medium
  • FCS phosphate buffered saline
  • FGF-2 FGF-2 from 0.01 to IOOng/ml was made up in the 10% serum containing media and 200 microlitres was added.
  • a TCP well had media containing no FGF-2 as. a control.
  • An initial cell count was Carried out and then further counts a 1, 2 and 4 days. By 4 days some of the ells were close to being confluent and so the study was stopped at this time point.
  • FIG. 8 shows the increased proliferative effect of FGF-2 in increasing concentrations on its addition to the media.
  • This experiment sho wed that 5000 cells used in initial attachment where confluent by 4 days, so the later experiments were seeded at 1000 HDFs/well.
  • Heparin Sigma Aldrich was incubated at 10 ⁇ / ⁇ 1 (200 ⁇ 1) in PBS for 18 hours, the heparin was then removed and wells washed three times in PBS.
  • FGF-2 was incubated at lOng/ml in PBS (200 ⁇ 1), A concentration curve of FGF-2 in the media was also produced, which confirmed that iOng/mt was sufficient to increase cell proliferation (Rusnati et al. 1 99, Zhu et a]. 2010), The same amount of FGF-2 was also incubated with wells modified with plasma polymer only (no heparin added). After 4 hours the FGF-2 was removed and the surfaces washed 3 times with. PBS.
  • HDFs human dermal fibroblasts
  • DMEM Dulbecco ' s Modified Eagles Medium
  • AA allylamine plasma polymer only
  • HFB AA+heparin and FGF-2
  • FB AA and FGF-2
  • Extra AA wells were also seeded so that heparin+FGF-2 (HFM) and FGF-2 alone (FM) alone could be added as a media supplement after seeding.
  • a tissue culture plate (TCP) was also seeded as a control. The ceils were left for 30 minutes post seeding to allow attachment then trie media was removed and the cells washed 3 times with PBS .
  • the number of cells per field of view is much lower than at higher serum concentration. As fewer cells am present after each reduction in serum condition, this indicates other important biomolecules for cell growth are being diluted out. Therefore, although the data shows that the GF is protected and active for longer periods when bound to the surface, a defined media would be required to achieve full potential in reduced and serum free environments.
  • the response seen at 2.5% and 1.25% serum could be for a number of reasons. Firstly as described above, the GF bound to the heparin may have a longer half life than GF added to the media, even when heparin is present because of the inhibiting effect of other proteins. This would limit protective effect of heparin on the GF which could then be degraded by enzymes present in the serum or produced by cells. There is also a lag at 1 and 2 days where cells appear not to be growing. This could be due to damage caused to the cell surface receptors when they undergo trypskiisation for seeding.
  • Bound GF would be advantageous at 1,25% and 2.5% serum conditions. This could mea a reduction in cost, as supplementing with GF would be required less frequently.
  • the data for 1.25% and 2,5% serum also confirms that there are other biomoleeules in the media which are important for cell viability.
  • K5P-2 on its own is not a total replacement for serum and other biotnoieeules could either be bound alongside PGF-2 to combat this or added via a defined media.
  • the modified surfaces could be used for growing sheets of dermal cells for grafting onto wounds.
  • patient Cells are harvested and grown in their own serum to avoid contamination and rejection of the grafted skin sheet (Munirah et al 2008; Schiestlef dl 201 1).
  • These modified surfaces could be used to increase the HDFs proliferation reduce serum use and speed up the growth of the -dermal grafts, which are often required urgently.
  • the same surface modification could also be incorporated into electrospun polymer scaffolds (Sun et al 2005) to mimic the ECM, which contains pools of GAGs and GFs. These scaffolds could then be used to produce a synthetic skin model to aid research into wound healing. This may reduce the need for animal testing and help reduce the limiting factors of skin donation from patients undergoing surgery.
  • a porous polymer scaffold is fabricated by 3D melt electrospinning of potycaproiactone. The scaffold is then treated with an amine and GAG as desribed herein. The scaffold is seeded with a mixed population of human primary fibroblasts and keratinooytes. This approach not only reduces the production costs/time associated with; the fabrication of a cell-seeded tissue engineered construct, but also aids cell self-organisation. Fibroblasts populate the interior of the scaffold, and keratinocyfes the exterior forming a primitive dermal-epidermal junction,
  • the PCL melt was then eiectrospuh using a collector distance Of 15 mtn, and flow rate of 20 ⁇ /h with a voltage of 10 kV applied to the spinneret.
  • Writing with the PCL fibers was achieved by collecting on a grounded plate connected to a programmable x-y stage, controlled using Mach 3 software.
  • the scaffold design consisted of interlaced diagonal lines at a traiislational velocity of 12.5 x !0 "3 rn/s,
  • FIG. 13a comprises a cylindrical stainless steel vacuum vessel with a diameter of 30cm and a volume of around 20 litres.
  • the scaffolds were suspended from a wire (Figure 13hj to: aid the plasma coating throughout the scaffold.
  • Figure 13c A schematic explaining how the scaffold was prepared for ToF-SMs analysis is shown in Figure 13c, The reactor was evacuated to a base pressure better than 4x10 "4 mbar, using a two- stage rotary pump with a liquid 2 cold trap. A needle valve was used to control the flow of monomer vapour into the reaction chamber.
  • Aliyiamine (AA) monomer (Sigma Aldrieh) underwent several freeze-pump-thaw cycles prior to use to remove dissolved gases. A total monomer flow rate of ca.
  • PCL unmodified, PCL ppAA coated, and PCL ppAA-heparin incubated scaffolds were all cross sectioned and analysed by time-of-flight secondary ion mass spectrometry ⁇ Tol?-SIMS).
  • a solution of heparin was made up at 10 flg/ml in PBS and 100 ⁇ was added per well to a 96 well plate to give 1 fig/well. The plate was incubated with a lid to prevent evaporation for 18 hours protected from light at room temperature, and was then washed twice with 130 ⁇ of PBS and dried on a paper towel. 150 ⁇ A of PBS-BSA 2% blocking solution was added to each well to prevent non-specific binding of proteins and antibodies in later steps, and incubated at 37°C for 1 hour and then washed .1 times in PBS.
  • FGF-2 basic fibroblast growth factor (R&D systems cat: 450-14) curve was made up starting with the highest concentration of 100 ng/well in 1 ⁇ ⁇ to the lowest which was given by 6 double dilutions, to 1.56 ng/well of FGF-2 and incubated at 37"C for 2 hours.
  • the epidermis was peeled from the dermis and proliferative keratinocytes scraped from the dermal-epidermal junction using a scalpel.
  • the ceils were een fuged for 5 minutes at 1200 rpm, resuspended in Greens media, and seeded onto irradiated 3X3 fibroblasts for expansion.
  • Keratinocytes were cultured in a 5% C ⁇ 3 ⁇ 4 incubator at 37° C and 95% humidity. Media was changed twice a week.
  • DMBM Duibeceo's Modified Eagles Medium
  • DMBM Duibeceo's Modified Eagles Medium
  • penicillin 100ru/ml
  • streptomycin 100 ug/ml
  • Scaffolds (either unmodified or with varying degrees of modification, denoted by conditions U 2 or 3, below) were seeded with cells. These variations were unmodified PCI. (I), PCL + ppAA (2) and PCL + ppAA + Heparin + FGF-2 (3). Scaffolds were placed in an untreated 6 well plate at the centre of each well and a seeding ring of 1 cm internal diameter was placed on top. 1ml of Greens media was placed around the scaffold to minimize cell loss from the scaffold. 4 x 10 5 fibroblasts (passage 3-6) were seeded iii 200/11 of Greens media into the centre of the rings arid allowed to. attach for 30 minutes. The centre Of the ring was topped up with media.
  • the ceils were allowed to attach for 30 minutes and then the well was topped up to 3 ml of Greens media once more.
  • the cells were maintained in culture for 4 days after which the seeding ring was removed and the scaffolds were raised to ah air liquid interface on a metal support grid in a fresh 6 well plate. Culture continued for a further 3 weeks with media changes every 2 to 3 days.
  • Resazurin sodium salt (Sigma) was dissolved in PBS at a concentration of 110 /tg/ml and filter sterilised to form a stock solution.
  • 10% (v/V) resazurin slock solution in cell culture medium forms the working solution.
  • the media was removed from the wells and the scaffolds moved to a new 24 well plate for the initial HDF attachment investigations. 500 ⁇ of the 10% resazurin solution was added to each well plus one blank well.
  • the assay was carried out in a 6 well plate with the seeding ring in place and 3 ml of the 10% (v/v) resazurin stock was required, in both cases the cells were incubated for 2 hours at 37 fl C in a 5% C ⁇ 3 ⁇ 4 atmosphere. At this point 200 ⁇ of the solution was removed to a 96 well plate and the fluorescence read at 544 nni on plate reader (BMG Labtech, UK). For the latter culture, the 3 ml of resazurin solution was removed to separate tubes and then mixed before 200 ⁇ of the solution as removed to a 96 well plat e.
  • Samples were then incubated with DAPI (1:100) and anti-pan-cytokeratra (1 :50) for 1 hr at room temperature whilst being protected from light, followed by washing 3 times with water to remove any salt crystals, and mounted. Samples were imaged using a Nikon AR confocal microscope.
  • the fiber diameter and pore size (interfiber distance) of the scaffolds were determined using image analysis software and found to range from 21 to 43 ⁇ with an average of 30.6 ⁇ 3.7 ⁇ (mean ⁇ 1 S.D.) for the fiber diameter, and 18.8 to 147,2 ⁇ , with an average of 67.8 ⁇ 27.8 ⁇ (mean ⁇ 1 S.D.) for the interfiber distance ( Figure I4a-c).
  • the deposited film contained only carbon and nitrogen elements present in the monomer with the addition of some oxygen (4%) ( Figure 21a-b).
  • the presence of oxygen in deposits from amine monomers is common, and is the result of either residual air and water within the reactor chamber, or the reaction of trapped radicals in the plasma polymer film with atmospheric oxygen and water.
  • the surfaces produced and used in this work contain similar chemistries to those previously show to: contain positive charges and bind GAG* in functional orientation (Robinson et al., 2012; Salim et al conflict 2009).
  • Figure 16a-c shows high resolution negative mass fragment peaks acquired in the region around 32 mix where S " (31,97 arnu) can be observed (in addition to (31.99 arou)). The intensity of S " fragment was very low in both mass spectra collected from PCL ( Figure 16a) and after modification with p AA ( Figure 16b).
  • FIG. 18 Viability data of fibroblasts on scaffolds are shown in Figure 18 conditions 1, 2 and 3. 4 x 10' fibroblast cells were seeded to investigate whether the modifications improved initial cell attachment to the scaffolds.
  • Figure 18 shows the results for resazurih assay on cells retained on the scaffold after 1 hour incubation. The results indicated that more viable cells were retained following successive modifications, and that there was an increase in cell viability for condition 2 and 3 compared to condition 1 (P ⁇ 0.05). Cells that had fallen through the scaffold to the well bottom during incubation were trypsinised off and counted to find seeding efficiency.
  • the data in Figure 19 shows the results of a resazurin viability assay after 2 weeks of culture of HDFs on the scaffolds under all 3 conditions. Results showed that modification of the scaffold increased cell proliferation, over the culture period, The ppAA (condition 2) scaffold was significantly different at (P ⁇ 0.05) from the unmodified scaffold (condition 1). Additional modification with heparin and FGF-2 (conditio 3) further increased HDF proliferation (P ⁇ 0.01 ).
  • Figure 20 shows ⁇ & ⁇ staining and fluorescent staining after co-culroring primary keralinocyte cells with the modified scaffolds following 2 weeks of culture with HDFs. Scaffolds were fixed and cryo-sectiotied as described above at 1, 2 and 3 weeks after the addition of keratinocyte cells. The histological stains indicated that there was a clear separation of cells into two layers. The H&E staining showed that the modified scaffolds improved cell organisation. It appeared that there were fewer fibroblasts stained only blue i the unmodified PCL scaffold. This would be expected because further modifications improved HDF cell viability and proliferation, infilling the: scaffold more rapidly oyer the 2 week culture period.
  • Plasma reactors are used experimentally in many fields of biomaterials research
  • GAGs can be passively adsorbed. Previously it. has been shown that passively boiind GAGs can bind proteins such as
  • Tissue Inhibitor of MetaUoproteinase 4 (TIMP-4). Recently published data has also demonstrated that FGF-2 can be bound functionally to adsorbed heparin and can subsequently affect cell proliferation over 8 longer tirne frame than when added to solution (Robinson, 2014; Robinson et al, 2012; Marson et at., 2009).
  • HLB sterilised acellular dermis
  • a further advantage is that this approach could be used to produce tailored eeil-seeded scaffolds where there is a potential to form two, or more, distinct cell layers.
  • HDF cells are seeded two- weeks prior to adding keratinocytes to the culture. It is likely the fibroblasts provide a good platform for the keratinocyte attachment with fewer falling through the scaffold.
  • fibroblasts produce many biomolecules that aid keratinocyte culture such as GFs and GAGs ( Yun at al., 2010) .
  • GFs and GAGs Yun at al., 2010
  • Plasma polymerisation has been used to increase the hioaetivity of synthetic tissue scaffolds.
  • Surface analysis showed that a uniform amine coatin is deposited by plasma throughput the scaffold. Scaffold surfaces were shown to bind heparin and FGF-2 (the functionality of which was confirmed by ELISA), and based upon comparison with controls it is evident that these scaffolds aid cell proliferation and the formation of distinct fibroblast and kerafmocyte layers.
  • the three- dimensional TE skin material fabricated usin this approach may meet the requirements of a reproducible, well defined.
  • the scaffolds described herein could be utilised to enhance current FJSE models as well as improve current commercially available products for treating chronic wounds. More broadly, this method of modification could be used to enhance the function of other synthetic scaffold materials using biomolecules, depending oh the application.
  • FaiTugia BL Dawson RA, Brown TD, Dalton PD, Hutmacher DW, Dargaville TR.
  • Salim M Wright PC, McArthur SL. Studies of electroosmotic flow and the effects of protein adsorption in plasma-polymerized microehannel surfaces. Electrophoresis. 2009;30:1877-87.
  • Skingmeering II transplantation flarge-scale laboratory-grown skin analogues in a new pig model. Pediatr Surg Int. 201 1 ;27:249-54.
  • FGF-7 Keratinocyte Growth Factor
  • Wainwright DJ Use of an acellular allograft dermal matrix (Allodenn) in the
  • Glycosaininoglycaiis can influence fibroblast growth factor-2 mitogenicity without significant growth factor binding.

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Abstract

La présente invention concerne un procédé d'immobilisation d'un facteur de croissance sur un substrat. Le procédé comprend la formation d'une surface à fonctionnalité amine sur le substrat. La surface à fonctionnalité amine est ensuite mise en contact avec un glycosaminoglycane dans des conditions de liaison non covalente du glycosaminoglycane et forme une surface à fonctionnalité glycosaminoglycane. La surface à fonctionnalité glycosaminoglycane est ensuite mise en contact avec un facteur de croissance dans des conditions pour lier le facteur de croissance à la surface.
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* Cited by examiner, † Cited by third party
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WO2017017425A1 (fr) * 2015-07-24 2017-02-02 The University Of Sheffield Implant médical
EP3258978A4 (fr) * 2015-02-16 2018-10-31 CTM@CRC Ltd. Procédés et produits de délivrance de cellules

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MEADE, K.A. ET AL.: "Immobilization of heparan sulfate on electrospun meshes to support embryonic stem cell culture and differentiation", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 288, 22 February 2013 (2013-02-22), pages 5530 - 5538 *
ROBINSON, D.E. ET AL.: "Glycosaminoglycan (GAG) binding surfaces for characterizing GAG-protein interactions", BIOMATERIALS, vol. 33, 2012, pages 1007 - 1016, XP028115478, DOI: doi:10.1016/j.biomaterials.2011.10.042 *
ZUBER, A.A. ET AL.: "Development of a surface to increase retinal pigment epithelial cell (ARPE-19) proliferation under reduced serum conditions", JOURNAL OF MATERIALS SCIENCE . MATERIALS IN MEDICINE, vol. 25, 2014, pages 1367 - 1373 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3258978A4 (fr) * 2015-02-16 2018-10-31 CTM@CRC Ltd. Procédés et produits de délivrance de cellules
WO2017017425A1 (fr) * 2015-07-24 2017-02-02 The University Of Sheffield Implant médical
US20180214613A1 (en) * 2015-07-24 2018-08-02 The University Of Sheffield Medical implant
US10765779B2 (en) 2015-07-24 2020-09-08 The University Of Sheffield Medical implant

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