WO2009105820A1 - Hydrogels dérivés de polymères biologiques - Google Patents

Hydrogels dérivés de polymères biologiques Download PDF

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Publication number
WO2009105820A1
WO2009105820A1 PCT/AU2009/000223 AU2009000223W WO2009105820A1 WO 2009105820 A1 WO2009105820 A1 WO 2009105820A1 AU 2009000223 W AU2009000223 W AU 2009000223W WO 2009105820 A1 WO2009105820 A1 WO 2009105820A1
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Prior art keywords
hydrogel
elastin
liquid phase
hydrogels
cross
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PCT/AU2009/000223
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English (en)
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Fariba Dehghani
Anthony Steven Weiss
Nasim Annabi
Suzanne Marie Mithieux
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The University Of Sydney
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Priority claimed from AU2008901006A external-priority patent/AU2008901006A0/en
Application filed by The University Of Sydney filed Critical The University Of Sydney
Publication of WO2009105820A1 publication Critical patent/WO2009105820A1/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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • 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
    • A61L27/56—Porous materials, e.g. foams or sponges

Definitions

  • the invention relates to biocompatible hydrogels.
  • a hydrogel is a network of polymer chains that are water-insoluble. They are superabsorbent (they can contain over 99% water) and they may be porous. Hydrogels may possess a degree of flexibility very similar to natural tissue due to significant water content.
  • hydrogels These characteristics mean that a number of laboratory and therapeutic applications have been anticipated for hydrogels. These include applications as scaffolds for tissue engineering, sustained release delivery systems, provision of articular surfaces and as dressing for wounds.
  • hydrogels The porous structure of hydrogels is anticipated to be particularly important in some of these applications such as tissue engineering and sustained release systems.
  • These hydrogels have generally been formed from synthetic polymers or natural polymers such as agarose and methylcellulose.
  • hydrogels are usually in contact with living tissue. This contact demands that the polymer chains be biocompatible with the tissue.
  • the invention seeks to at least minimise one or more of the above problems or limitations and in one embodiment provides a method for producing a hydrogel.
  • the method includes:
  • a method for producing a hydrogel including one or more pores includes:
  • an apparatus for forming a hydrogel including:
  • a vessel including a liquid phase, the liquid phase including coacervated elastin material;
  • - pressurising means for providing the vessel with a gas to pressurise the vessel; - injection means for injection of a cross linking agent for cross linking the coacervated elastin material into the liquid phase; and optionally
  • - input means for input of one or more components to be provided in a hydrogel formed by the apparatus, the one or more components selected from the group consisting of a protein, a sugar, a lipid, a cell and a pharmaceutical.
  • hydrogel including:
  • the hydrogel characterised in that the scaffold of cross linked elastin material molecules are arranged to provide the hydrogel with pores that extend throughout the hydrogel.
  • hydrogel produced by a method according to one of the embodiments described above.
  • Figure 1 shows a schematic diagram of an apparatus of one of the embodiments of the invention, which is a one-step process for simultaneous cross-linking, homogenous pore formation in the polymer matrices and removing residual of cross-linking agent.
  • Figure 2 shows a schematic diagram of an apparatus of another of the embodiments of the invention.
  • Figure 3 depicts the effect of CO 2 pressure on coacervation temperature of ⁇ -elastin
  • Figure 4 depicts effect of CO 2 pressure on the coacervation of ⁇ -elastin at 37 0 C: control ( ⁇ ), 180 bar( # )
  • Figure 5 depicts a table presenting the effect of CO 2 pressure on the time to achieve maximum coacervation for ⁇ -elastin.
  • Figure 6 presents SEM images of ⁇ -elastin hydrogel (a) and(b) at 100 bar; (c) and (d) at 60 bar; (e) and (f) at 150 bar; (g) at 60 bar and (h) at 1 bar.
  • Figure 7 presents a skyscan analysis of lyophilised ⁇ -elastin hydrogels fabricated at 100 bar (a) and 1 bar (b)
  • Figure 8 shows images of fibroblast cells cultured on hydrogels produced at 60 bar CO 2 (a, b) and atmospheric pressure (c)
  • Figure 9 demonstrates the effect of media on swelling ratio of ⁇ -elastin hydrogels provided in Example 4 at 4 0 C (0.5% (v/v) GA).
  • Figure 10 depicts (a) ESEM image of a wet ⁇ -elastin hydrogel produced at 60 bar CO 2 pressure. SEM images of an ⁇ -elastin hydrogel fabricated at (b) 60 bar CO 2 pressure, (c) 100 bar CO 2 pressure and (d) atmospheric pressure. Note that figure 10 (d) and 6(h) are equivalent; the figure is presented again for comparative purposes.
  • Figure 11 presents a skyscan analysis of lyophilised ⁇ -elastin hydrogels fabricated at high pressure CO 2 (a) and 1 bar (b). Note that figure 11(b) and 7(b) are equivalent; the figure is presented again for comparative purposes.
  • Figure 12 depicts SEM images of fibroblast cells attached to an ⁇ -elastin hydrogel fabricated by high pressure CO 2 using 0.5 % (v/v) GA (a) top surface, (b) to (g) internal surface of channel obtained by cross sectioning the sample, (h) control sample - an unseeded hydrogel. Sheets of cells can be seen in images (a)-(d) and individual cells in images (e)-(g).
  • Figure 13 depicts SEM images of ⁇ -elastin hydrogels fabricated at (a, b) atmospheric pressure using 2 % HMDI, (c, d) 60 bar CO 2 pressure using 2 % HMDI, (e, f) 60 bar CO 2 pressure using 5 % HMDI, and (g) 100 bar CO 2 pressure using 2 % HMDI.
  • Figure 14 depicts unconfined compressive behaviour of HMDI crosslinked hydrogel. Cyclic stress-strain data for the sample produced at high pressure CO 2 (a) and atmospheric condition (b). Compressive modulus (c) and energy loss (d) at each strain level for both hydrogels produced at high pressure and atmospheric condition.
  • Figure 15 depicts the swelling behaviour of fabricated hydrogel produced at high pressure and atmospheric condition in PBS, water, and DMSO.
  • Figure 16 depicts the relationship between the swelling ratio in PBS and compressive modulus for fabricated hydrogels at high pressure CO 2 and atmospheric condition.
  • Figure 17 depicts images of fibroblast cells cultured on (a-c) hydrogel produced at 60 bar CO 2 and (d and e) atmospheric pressure.
  • Figure 18 depicts SEM images of fibroblast cells attached to ⁇ r-elastin hydrogel fabricated at atmospheric condition (a), and high pressure CO 2 (b-f).
  • (a), (b) and (c) depict the top surface of the hydrogel whilst (d) to (f) depict internal surfaces of hydrogel obtained by cross sectioning the sample.
  • Figure 19 depicts the swelling behaviour of elastin-tropoelastin hydrogels produced at high pressure CO 2 and atmospheric condition in PBS at 37°C.
  • Figure 20 depicts SEM images of TPE/ ⁇ -elastin hydrogels generated at atmospheric pressure using (a) 0.1 , (b) 0.25, and (c) 0.5 % (v/v) GA.
  • Figure 21 depicts SEM images of TPE/ ⁇ -elastin hydrogels fabricated at (a-d) 60 bar CO 2 pressure, (e-h) atmospheric pressure. Top surface of the samples are shown in images (a), (b), (g), and (h), cross sections in images (c)-(f).
  • Elastin is a molecule that is found in many tissues. It is essentially formed by a two step process, the first involving an alignment of tropoelastin or elastin fragments that brings functional groups into close proximity through an interaction of hydrophobic groups known as "coacervation". The second step involves the cross linking of aligned or "coacervated” elastin so as to form covalent bonds between the aligned relevant functional groups.
  • Cross linking is generally achieved in a living system by lysyl oxidase. In the laboratory this may be achieved using a range of reagents including glutaraldehyde, amine-reactive chemical crosslinkers including BS3 and amine and carboxyl reactive crosslinkers including EDC.
  • Coacervation is a critical step. Without this step it is basically not possible to effect a cross linking reaction which would produce elastin material in the form as is generally observed in a living system. Coacervation is a reversible step - molecules can be "unaligned" by manipulating pH, temperature or salt. The effect of pressure on coacervation at the time of this invention was basically not known.
  • the inventors have found that while pressure does affect coacervation, it is nonetheless possible to coacervate elastin materials where an elastin material would include tropoelastin, purified elastin such as ⁇ elastin, sub-fragments or elastin like peptides under pressures greater than normal atmospheric pressure.
  • the inventors have recognised one application which is that hydrogels, in particular porous hydrogels can be generated by applying pressure to an elastin material coacervate together with cross linking. This enables the formation of hydrogels having biocompatible molecules therein and which may have a porous structure.
  • a method for producing a hydrogel includes:
  • Hydrogel generally refers to a substance formed from, or comprised of, a network of polymer chains that are water-insoluble, in which water is the dispersion medium. Hydrogels are superabsorbent (they can contain over 99% water). Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content.
  • the vessel including the liquid phase having coacervated elastin material may be provided by initial steps including: -providing a vessel including a liquid phase, the liquid phase in the form of a solution of elastin material and
  • the liquid phase may be provided with a cross linking agent for cross linking elastin material in the liquid phase before or after the vessel is pressurised, or after it is depressurised.
  • liquid phase is provided with a cross linking agent to cross link coacervated elastin material in the liquid phase before the vessel is pressurised.
  • the liquid phase is provided with a cross linking agent to cross link coacervated elastin material in the liquid phase after the vessel has been pressurised.
  • cross linking can occur either at maintained pressurisation or after some of the pressure has been released.
  • the hydrogel formed after depressurisation is provided with one or more pores. These pores are particularly useful for increasing the water binding capacity of the hydrogel. They are also useful for providing a surface for providing the hydrogel with other components such as pharmaceutical drugs, structural and bioactive proteins, sugars and lipids. In certain embodiment the pores are useful for seeding with cells such as fibroblasts and the like.
  • cross linking of coacervated elastin material after gas has been dissolved in the liquid phase tends to entrap dissolved gas.
  • the release of dissolved gas from the cross linked elastin material as the vessel is depressurised is believed to form pores in the cross linked elastin material, hence providing a hydrogel having pores.
  • One particular advantage of this embodiment of the invention is that it is possible to control pore size, shape and distribution throughout the hydrogel formed by the process by controlling the amount of pressure provided to the vessel, the rate of depressurisation, the concentration of cross linking agent and coacervated elastin material.
  • the step of cross linking the coacervated elastin material after pressurisation of the vessel entraps gas molecules dissolved in the liquid phase within the cross linked elastin material.
  • the step of depressurisation provides conditions for release of gas entrapped within the cross linked elastin material.
  • the release of gas entrapped within the cross linked elastin material provides the hydrogel formed from the cross linked elastin material with one or more pores.
  • the one or more pores preferably form one or more conduits that extend throughout the hydrogel or ramify to form networks throughout the hydrogel.
  • the cross linking agents may be enzymatic (such as lysyl oxidase) or chemical, examples of the latter being glutaraldehyde (GA), amine-reactive chemical cross linkers and amine and carboxyl reactive cross linkers.
  • amine-reactive cross linkers include disuccinimidyl glutarate (DSG), bis(sulfosuccinimidyl) suberate (BS3), ethylene glycol diglycidyl ether (EGDE) under neutral conditions (pH 7), hexamethylene diisocyanate (HMDI), tris-succinimidyl aminotriacetate (TSAT), disuccinimidyl suberate (DSS) and ⁇ -[tris(hydroxymethyl)phosphino]propionic acid (THPP).
  • DSG disuccinimidyl glutarate
  • BS3 bis(sulfosuccinimidyl) suberate
  • EGDE ethylene glycol diglycidyl
  • carboxyl reactive cross linkers examples include1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), and ethylene glycol diglycidyl ether (EGDE) under acidic conditions (pH ⁇ 4).
  • EDC 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
  • EGDE ethylene glycol diglycidyl ether
  • the cross linker may be provided in the liquid phase in an amount of from about 0.05 to about 10 percent (v/v). In one embodiment, the cross linker is GA and is provided in an amount of from about 0.05 to about 5 percent (v/v). In another embodiment the cross linker is GA and is provided in an amount of from 0.05 to about 2 percent (v/v). In another embodiment the GA is provided in the liquid phase in an amount of from about 0.1 to about 0.5 percent (v/v).
  • the cross linker is HMDI and is provided in the liquid phase in an amount of from about 0.25 to about 10 percent (v/v). In another embodiment, HMDI is provided in the liquid phase in an amount of from about 1 to about 5 percent (v/v). In another embodiment, the HMDI is provided in the liquid phase in an amount of from about 2 to about 5 percent (v/v).
  • the conditions provided to the vessel for coacervation of elastin material in the solution may be provided at the time that the vessel is pressurised with a gas to dissolve the gas into the liquid phase.
  • the conditions for coacervation may be provided before pressurisation of the vessel so that at the time of pressurisation the elastin material contained in the liquid phase is presented in the form of a coacervate.
  • elastin material for use in producing the hydrogel may be cross linked before coacervation.
  • An example of such an elastin material is one obtained by extraction from a tissue source, such as from bovine tissue, ⁇ -elastin is one particular example.
  • the elastin material may not be cross linked at all before coacervation.
  • One example of such a molecule is tropoelastin.
  • ELP elastin like peptide
  • the elastin material is selected from the group consisting of tropoelastin, an elastin such as ⁇ -elastin, sub-fragments of these molecules and ELP.
  • the elastin material has been obtained by extraction from a tissue.
  • the elastin material has been obtained from a recombinant expression system, examples of which are discussed in WO99/03886, WOOO/04043 and WO94/14958, the contents of which are disclosed herein in their entirety by reference, or from peptide synthesis such as solid phase peptide synthesis.
  • Elastin may be provided in the liquid phase in a range of concentrations. In one embodiment, the elastin is provided in the liquid phase in an amount of from about 5 to about 200 mg/mL.
  • the liquid phase may further include one or more further biomolecules.
  • biomolecules include proteins, sugars and lipids.
  • the biomolecule is a protein
  • connective tissue proteins and extra cellular matrix proteins such as collagens and the like are particularly preferred.
  • Other bioactive proteins such as hormones, growth factors or cytokines are also useful.
  • the biomolecule is a sugar
  • molecules such as glycosaminoglycans are particularly preferred.
  • the liquid phase further includes a pharmaceutical compound.
  • a pharmaceutical compound examples include anti cancer compounds, such as anti angiogenic compounds and compounds useful for tissue repair or regeneration.
  • the pH of the liquid phase is generally selected to prevent coacervation from reversing to form soluble protein. Generally, coacervation may occur when the pH value of the liquid phase is between about pH 3 and 8.
  • the pH of the liquid phase may be influenced by the gas used to pressurise the vessel.
  • the liquid phase may include a buffer.
  • the liquid phase includes, or is phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the liquid phase may include a salt.
  • suitable salts NaCI, KCI and NH 4 CI.
  • the ionic strength of the liquid phase is generally about between about 0 and about 1.5 M.
  • the liquid phase is selected having regard to the solubility of the cross linking agent.
  • Aqueous solutions are preferred for cross linking agents that are soluble in water.
  • water or PBS can be used where the cross linker is glutaraldehyde.
  • Non aqueous solutions are preferred for agents that are not soluble in water.
  • DMSO can be used for HMDI cross linkers as discussed in the Examples herein.
  • Suitable non-aqueous liquid phases include solvents that can dissolve a dense gas such as CO 2 .
  • Other examples of non-aqueous solvents include ethanol, acetone, isopropanol, dimethyl formamide, and ethyl acetate.
  • the method generally includes a further step of removing the non aqueous liquid phase from the elastin material after depressurisation and contacting the elastin material with water to form the hydrogel.
  • the gas that is used to pressurise the vessel is generally any gas that is capable of dissolving into a liquid or otherwise an aqueous solution under pressure.
  • gases include CO 2 and N 2 gas.
  • Other examples include ethane, methane, 1 ,1 ,1 ,2- tetrafluoroethane, NO 2 , propane and heptane gas.
  • CO 2 gas is particularly useful.
  • the pressures range from about 10-300 bar. Most preferred pressures are within the range of from about 30-100 bar. Alternatively, when the gas is CO 2 , the preferred pressures are within the range of about 5 to about 180 bar.
  • the vessel is pressurised in conditions in which the gas remains below the critical point.
  • This state is sometimes referred to as a dense gas (DG). That is, the gas is not in a state generally recognised as a supercritical fluid.
  • the pressure to be provided is to be determined having regard to desired characteristics of the hydrogel to be produced according to the method.
  • One particular characteristic is the porosity of the hydrogel. In certain embodiments this is understood to be a function of the molar amount of gas dissolved into the liquid phase.
  • the pressure to be provided to the vessel dissolves an amount of CO 2 into the liquid phase, in case of aqueous solution effective for reducing the pH of the liquid phase.
  • the vessel is pressurised in conditions in which the gas is present as a supercritical fluid.
  • these embodiments may be advantageous where there is a need to coacervate at lower than room temperatures.
  • the inventors have surprisingly found that elastin can be made to coacervate at temperatures approaching 15 0 C when exposed to supercritical CO 2 .
  • conditions suitable for the provision of supercritical fluid include pressures of at least about 73.8 bar and temperatures of at least about 31 0 C.
  • the method further includes the step of separating the liquid phase from the hydrogel.
  • the method includes the step of desiccating the hydrogel to form a dried form of the hydrogel. This may be then be sold as a dried form to which water may be added to reconstitute the hydrogel.
  • the method includes the further step of providing the hydrogel formed by the method with a pharmaceutical compound. Generally this can be achieved by methods known in the art. In yet further embodiments, the method includes the further step of providing the hydrogel with a cell such as a fibroblast or stem cell.
  • an apparatus for forming a hydrogel including:
  • - pressurising means for providing the vessel with a gas to pressurise the vessel
  • - input means for input of one or more components to be provided in a hydrogel formed by the apparatus, the one or more components selected from the group consisting of a protein, a sugar, a lipid, a cell and a pharmaceutical.
  • the injection means is adapted to automatically inject the cross linking agent when the vessel reaches a pre-selected pressure.
  • An ⁇ -elastin solution is pipetted into the sample holder placed in the temperature controlled high pressure vessel 7.
  • the high pressure pump 4 is then pressurised with liquid CO 2 by opening valves 2 and 3.
  • Valves 5 and 6 are opened when the vessel approaches thermal equilibrium.
  • Vessel 7 is then slowly pressurised by CO 2 while valve 10 is closed.
  • the temperature of the vessel is kept constant using a temperature controller 8 during cross linking.
  • the system is isolated by shutting down valves 2, 3, 5 and 6 and maintained at constant temperature and pressure and then depressurised by opening valve 10. Pore formation in the biopolymer matrix may occur upon depressurisation and the residual cross linking agent can be removed by passing CO 2 through the sample by opening valves 2,3, 5,6 and running pump 4 at constant pressure mode.
  • the flow rate of CO 2 is controlled during this stage by valve 10 prior to depressurisation.
  • a hydrogel the hydrogel including:
  • the hydrogel characterised in that the scaffold of cross linked elastin material molecules are arranged to provide the hydrogel with pores that extend throughout the hydrogel.
  • the one or more pores form one or more conduits that extend throughout the hydrogel or ramify forming networks throughout the hydrogel.
  • the scaffold is preferably formed by cross linking tropoelastin.
  • hydrogel produced by a method according to one of the embodiments described above.
  • Elastin the major component of elastic fibers, is an insoluble extracellular matrix protein found in skin, bladder, lung, ligament, elastic cartilage and arteries. It provides elasticity and resilience to maintain the proper function of tissues that are subjected to repetitive distension and physical stress. Elastin is the most persistent protein in the body. It is particularly established by assembly of its precursor tropoelastin in utero.
  • Elastin is an extremely insoluble biopolymer and it is difficult to process into new biomaterials so ⁇ -elastin, an oxalic acid- solubilized derivation of elastin, is frequently used for synthesizing elastin-based materials as it undergoes reversible self-association.
  • ⁇ -elastin an oxalic acid- solubilized derivation of elastin
  • Coacervation plays a crucial role during elastin formation by concentrating and aligning monomers prior to crosslinking to form fibers. Coacervation of these soluble molecules is a concentration- dependent process and can be interpreted as an intermolecular hydrophobic association where solution variables such as biopolymer concentration, pH, salt and impurities can influence its efficacy.
  • ⁇ -elastin (molecular weight »60000) extracted from bovine ligament was purchased from Elastin Products Co. (Missouri USA). Carbon dioxide (99.99 % purity) and high purity nitrogen were supplied by BOC. All aqueous solutions were prepared in MiIIiQ water, ⁇ -elastin was dissolved in phosphate-buffered saline (10 mM sodium phosphate, 135 mM NaCI, pH 7.4; PBS) at 5 mg/mL and the solution was dissolved overnight at 4°C prior to use.
  • phosphate-buffered saline 10 mM sodium phosphate, 135 mM NaCI, pH 7.4; PBS
  • FIG. 2 The schematic diagram of the apparatus used for investigating the effect of CO 2 pressure on ⁇ -elastin is shown in Figure 2.
  • a high pressure pump (Thar, Model P50) was used to transfer CO 2 into the high pressure view cell (Jerguson sight gauge, Model R32).
  • a recirculation heater (Ratek) was used to control the temperature of the water bath and the pressure of the system was monitored using a pressure transducer (Druck).
  • the coacervation temperature of ⁇ -elastin is governed by various factors including pH, ionic strength, and protein concentration.
  • the coacervation temperature of tropoelastin and ⁇ -elastin decreased as the ionic strength increased.
  • the influence of pH on coacervation temperature of ⁇ -elastin dissolved in pure water, in buffer solution with high and low ionic strength, and tropoelastin solution at PBS (150 mM NaCI) were studied.
  • the highest level of absorption and the lowest coacervation temperature were observed.
  • the coacervation temperature increased and the rate of turbidity decreased. Coacervation was prevented at pH values far from the isoelectric point of ⁇ -elastin at below 3 or above 8.
  • the effect of DG CO 2 on coacervation temperature and partially reversible coacervation of ⁇ -elastin solution may be due to the lower free energy of the hydrophobic core of the coacervate, compared with the usual conformation, which then achieved a folded state.
  • the reverse transition is, therefore, no longer thermodynamically favorable and the newly formed coacervate might have a higher stability.
  • the main contributing factor to this transition and exact mechanism of action is currently unknown.
  • the reduction in the coacervation temperature of ⁇ -elastin solution in DG CO 2 system may be due to a decrease in the pH of the protein solution caused by dissolved CO 2 with a potential contribution by a depression of the melting temperature of the hydrophobic core caused by the pressure.
  • Each of these factors has the potential to lower the activation energy of the transition by inhibiting unfavorable side chain interactions that form spontaneously during the folding process and present distinctive energy barriers that have to be overcome in order to achieve a functional final set of conformations.
  • High pressure CO 2 is volatile and can dissolve in aqueous solutions to decrease the pH due to the acidification of aqueous solution and production of carbonic acid.
  • solubility of CO 2 in water is a function of temperature and pressure, the drop in pH of the solution will be temperature and pressure dependant.
  • a decrease in pH of the ⁇ - elastin solutions exposed to CO 2 may lead to a decrease in coacervation temperature of ⁇ -elastin.
  • the partial reversible coacervation of a- elastin in DG CO 2 system may result from both the pH drop in the system caused by dissolved CO 2 and the interaction between the CO 2 and ⁇ -elastin while the retardation in the coacervation profile of ⁇ -elastin solution exposed to CO 2 may be associated with the interaction between CO 2 and ⁇ -elastin that may take longer time to approach the initial condition.
  • a buffer solution containing ⁇ -elastin and the cross-linking agent glutaraldehyde was prepared. The solution was then placed in a high pressure vessel and the system was connected to the high pressure pump. After purging the air from the system and thermal equilibrium the system was pressurized slowly. The sample was kept at the desired pressure and temperature for a defined time and then the system was depressurised in a controlled manner.
  • the following microscopic images ( Figure 6) compare the characteristics of hydrogels fabricated by the process described by this invention and the samples produced at atmospheric conditions.
  • the ⁇ -elastin hydrogels were formed at atmospheric pressure using 100 mg/mL of ⁇ - elastin and 0.5 % GA and incubating the sample at 37°C for one hour.
  • MG 3348 cells were maintained in Advanced Dulbecoo's Modified Eagle's Medium (DMEM) supplemented with 12.5 ml/liter antibiotic-antimycotic, 12.5 ml/liter L-glutamine (Invitrogen, Australia) and 5% v/v fetal bovine serum (FBS) (JRH biosciences, Kansas, USA). TrypLETM express stable trypsin-like enzyme was purchased from Invitrogen. 24- well-plates were obtained from Costar Corp, Cambridge, MA.
  • DMEM Advanced Dulbecoo's Modified Eagle's Medium
  • FBS 5% v/v fetal bovine serum
  • fibroblast cells (MG 3348) cultured on GA cross-linked ⁇ -elastin hydrogels, fabricated by the process disclosed in this invention and at atmospheric pressure, was studied. Prior to seeding the cells on fabricated scaffolds, the cells were grown in DMEM media containing 5% FBS for 3 weeks to obtain sufficient cells for seeding on scaffolds. Fibroblast cells (MG 3348) were plated on 25-cm 2 flasks containing 12 ml_ DMEM media with 5% FBS. The growth of cells in the media was confirmed using an optical microscope after which the cells were transferred into a 75 cm 2 flask).
  • the hydrogels were transferred into individual wells of 24-well plates, washed twice with ethanol and subsequently with cell culture media.
  • the ⁇ -elastin scaffolds were equilibrated with culture media by soaking in 2 ml_ DMEM media containing 5% FBS at 37°C incubator overnight. The DMEM media were then replaced with 2 mL culture media containing cells.
  • the scaffolds that were seeded with cells were left for 2 days at 37°C in a CO 2 incubator. Cell proliferation was visually assessed by optical microscopy and SEM after fixing and staining seeded scaffolds.
  • the existence and growth of cells in fabricated hydrogels was confirmed using light microscopic analysis after fixing and staining of slides of cross-sections of seeded scaffolds to show cellular growth in fabricated scaffolds.
  • the cells were fixed into the scaffolds by soaking in 10% formalin and the scaffolds were immersed in 70% ethanol.
  • the samples were processed on an automated tissue processor on a 6 hour cycle to paraffin through a graded series of ethanol, and xylene. They were embedded in paraffin wax and 5 ⁇ m sections were taken and collected onto glass slides and dried.
  • the slides were then deparaffinised, rehydrated, stained using a standard haematoxylin and eosin staining procedure, dehydrated, cleared in xylene and mounted in DPX. Then, the slides were monitored using light microscope connected to the camera.
  • the cells could infiltrate through the channels in the hydrogel matrix.
  • the attachment of the cells to the hydrogel matrix corroborates the biocompatibility of fabricated hydrogels produced at high pressure CO 2 .
  • Engineering large channels for the cells to penetrate in the hydrogel matrix are the advantages of this novel method.
  • ⁇ -elastin extracted from bovine ligament was obtained from Elastin Products Co. (Missouri USA). All aqueous solutions were prepared in MiIIiQ water, ⁇ -elastin was dissolved in PBS (Phosphate-buffered saline) (10 mM sodium phosphate pH 7.4, 1.35 M NaCI). Gluteraldehyde (GA) was purchased from Sigma. Food grade carbon dioxide (99.99 % purity) was supplied by BOC. GM3348 cell line was obtained from the Coriell Cell Repository. Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS), penicillin and streptomycin. All tissue culture reagents were obtained from Sigma.
  • DMEM Dulbecco's Modified Eagle's Medium
  • ⁇ -elastin solution was mixed with GA and the solution was immediately pipetted into a custom-made Teflon mould. The mould was then placed at 37°C for a period of time ranging from 15 min to 24 hr, to fabricate a hydrogel.
  • the crosslinked hydrogels were washed in MiIIiQ water, then placed in 100 mM Tris ((HOCH 2 ) 3 CNH 2 ) in PBS for 1 hr to inhibit further cross-linking and stored in PBS for characterisation.
  • DGHF Dense Gas Hydrogel Formation process
  • FIG. 1 A schematic diagram of the apparatus used to fabricate ⁇ -elastin hydrogels using dense gas CO 2 is shown in Figure 1.
  • a syringe pump (ISCO, Model 500D) was used to transfer CO 2 into the high pressure vessel (Thar, 100 ml_ view cell).
  • the vessel was comprised of a temperature and pressure controller that allowed for temperature adjustments and the monitoring of both variables accurately.
  • a peristaltic pump (MHRE 200) was used for cold water recirculation in the jacket of the syringe pump to condense CO 2 into liquid when the pump was filled with CO 2 .
  • ⁇ -elastin solution containing the cross-linker was injected into a custom-made teflon mould placed inside the high pressure vessel. After the vessel was sealed and approached thermal equilibrium at a specific temperature, the system was pressurised with CO 2 to the desired level, isolated and maintained at these conditions for a set period of time. The system was then depressurised and samples were collected. Crosslinked structures were immediately washed repeatedly in MiIIiQ water, then placed in 100 mM Tris in PBS for 1 hr. After Tris treatment, the hydrogels were washed twice in MiIIiQ water and stored in PBS for further analysis.
  • the swelling property of hydrogels can be correlated to the degree of crosslinking through the hydrogel matrices.
  • the crosslinked hydrogels were placed in liquid nitrogen for 5 min and then lyophilised for 20 hr using a freeze dryer.
  • the swelling properties were measured at four different conditions, 37 0 C and 4 0 C using either PBS or MiIIiQ water. Under each set of conditions, at least three samples were placed in the media overnight. The excess liquid was then removed from the swollen samples and the swollen mass was recorded.
  • the swelling ratio was calculated using the following equation:
  • Lyophilized ⁇ -elastin hydrogels were analysed using a Skyscan 1072 (Skyscan, Belgium) high-resolution desktop X-ray CT scanner at 2.94 ⁇ m voxel resolution, X-ray tube current 160 ⁇ A and voltage 62 KV to obtain 3D reconstructed images.
  • the samples were mounted vertically on a plastic support and rotated through 360° around the z-axis of the sample. 3D reconstruction of the samples was carried out using axial bitmap images and analysed by VG Studio Max software (Volume Graphics GmbH, Heidelberg, Germany). Scanning electron microscopy (SEM)
  • the SEM images of samples were obtained using a Philips XL30 at 15 KV to determine the pore characteristics of the fabricated hydrogels and to examine cellular infiltration and adhesion.
  • Lyophilised ⁇ -elastin hydrogels were mounted on aluminium sample stubs using conductive carbon paint then gold coated prior to SEM analysis.
  • Cell- seeded hydrogels were fixed with 2% GA in 0.1 M Na-cacodylate buffer with 0.1 M sucrose for 1 hr at 37°C. Samples underwent post-fixation with 1 % osmium in 0.1 M Na- cacodylate for 1 hr and were then dehydrated in ethanol solutions at 70%, 80%, 90% and 3 times 100% for 10 min each.
  • HMDS hexamethyldisilazane
  • hydrogels were transferred into a 24- well plate and washed twice with ethanol to sterilise the materials. The hydrogels were then washed at least twice with culture media to remove any residual ethanol and equilibrated in culture media (DMEM, 10 % FBS, pen-strep) at 37 0 C over night. The cells were then seeded onto the hydrogels at 3 * 105 cells/well. An unseeded hydrogel was also kept in a well as a control sample. The cells were cultured in a CO2 incubator for 2 days at 37°C, after which the hydrogels were fixed to assess cell proliferation and infiltration using SEM analysis.
  • DMEM 10 % FBS, pen-strep
  • the growth of the cells in fabricated hydrogels was confirmed using light microscopic analysis after fixing, sectioning, and staining cross sections of cell seeded scaffold.
  • the hydrogels containing cells were fixed by soaking in 10% formalin over night.
  • the scaffolds were then immersed in 70% ethanol.
  • the samples were processed on an automated tissue processor on a 6 hour cycle to paraffin through a graded series of ethanol, and xylene. They were embedded in paraffin wax and 5 ⁇ m sections were taken and collected onto glass slides and dried.
  • the slides were then deparaffinised, rehydrated, stained using a standard haematoxylin and eosin staining procedure, dehydrated, cleared in xylene and mounted in DPX. Then, the cross-sections were examined a using light microscope connected to the camera.
  • ⁇ -elastin hydrogels fabricated in this study exhibited high swelling ratios, in the range of 21-35 g H 2 OZg protein, in water. Increasing the processing pressure from 30 bar to 150 bar resulted in a 60% increase in the hydrogel swelling ratio as indicated in Figure 9.
  • Both hydrogels produced at high pressure CO 2 and atmospheric pressure displayed stimuli-responsive characteristic toward temperature and salt concentrations when they were swelled in PBS and water at 4 C C and 37 0 C.
  • the swelling ratio of the hydrogels in water was greater than those swelled in PBS at 4 0 C as shown in Figure 9.
  • the fabricated hydrogels swelled more in both water and PBS at lower temperature as indicated in Table 2.
  • Hydrogels produced at 60 bar CO 2 pressure with 0.5% (v/v) GA absorbed 33.2 ⁇ 0.8 and 28.6 ⁇ 1.6 g liquid/g protein at 4 0 C and 37 0 C, respectively, when they were hydrated in water. However, they absorbed 18.3 ⁇ 4.6 and 7 ⁇ 3.2 g liquid/g protein at 4 0 C and 37 0 C, respectively, when they were swelled in PBS. Elevated temperature and the presence of salt in PBS resulted in a contraction of the material due to water expulsion. As exhibited in Table 2, the swelling behaviour of the GA crosslinked ⁇ -elastin hydrogels produced at high pressure CO 2 was either considerably greater or comparable with other elastin based hydrogels fabricated using various cross-linkers.
  • the variation of pressure and crosslinker concentration had a significant effect on the swelling behaviour of fabricated hydrogels.
  • the swelling ratio of fabricated hydrogels was enhanced by increasing the pressure and reduced by enhancing the crosslinker concentration.
  • the swelling ratio of the samples exposed to high pressure CO 2 was increased from 21.4 ⁇ 0.7 to 35.2 ⁇ 2.5 g H 2 O/ g protein as pressure increased from 30 bar to 150 bar at 0.5 % (v/v) GA concentration.
  • Using higher concentration of cross-linker resulted in higher degree of cross-linking through the hydrogel matrices which resulted in a reduction in the swelling ratio.
  • Table 3 Effect of pressure and crosslinker concentration on the swelling ratio of fabricate hydrogels at high pressure CO 2 .
  • Pore size and interconnectivity are critical hydrogel properties that influence the ability of cells to infiltrate and proliferate within 3D structures.
  • ESEM, SEM, and Micro-CT analysis were used to characterise the pore morphology of fabricated ⁇ - elastin hydrogels.
  • ESEM analysis demonstrated that ⁇ -elastin hydrogels fabricated under high pressure CO 2 ( Figure 10a) were highly porous yet robust structures.
  • ESEM images of ⁇ -elastin hydrogels produced under atmospheric pressure could not be obtained because the wet constructs disintegrated prior to examination.
  • the average pore size of hydrogels decreased from 14.3 ⁇ 3.3 ⁇ m to 4.9 ⁇ 4.3 ⁇ m as the pressure increased from 1 bar to 60 bar.
  • the pore wall thickness was dramatically diminished to 0.5 ⁇ 0.1 ⁇ m compared to 4.3 ⁇ 5.5 ⁇ m for ⁇ -elastin hydrogels fabricated under atmospheric conditions. This represents nearly a 10-fold reduction in pore wall thickness of ⁇ -elastin hydrogels fabricated at high pressure CO 2 compared with the samples produced at atmospheric pressure.
  • the organisation of the matrix produced under high pressure is similar to that seen in some natural elastin microstructures within the body [7, 8].
  • Cross-linked ⁇ -elastin hydrogels fabricated under atmospheric conditions consisted of polyhedral shaped pores with thick walls. However, highly interconnected pores with thin walls were formed when ⁇ -elastin was cross-linked under high pressure CO 2 .
  • the thickness of the walls between pores was also diminished from 4.3 ⁇ 5.5 ⁇ m to 0.5 ⁇ 0.1 ⁇ m and 0.46 ⁇ 0.11 ⁇ m when pressure increased from 1 bar to 60 bar and 100 bar.
  • dense gas CO 2 had no significant effect on the pore size of fabricated hydrogels.
  • a unique feature of cross-linking elastin under high pressure CO 2 is the formation of micro-channels throughout the 3D structure of the hydrogels as demonstrated in Figure 6(d) and Figure 6(g). These channels were most likely induced during the depressurisation stage.
  • the size of the pores fabricated at both atmospheric and high pressure CO 2 were smaller than 15 ⁇ m as indicated in Figure 10. Whilst these pore sizes are suitable for the diffusion of nutrients, oxygen, and waste from cells they are not large enough to allow for cells such as fibroblast to penetrate and grow into the matrices.
  • the presence of communicating larger channels with dimensions of 97 ⁇ 21.4 ⁇ m was expected to allow for cell infiltration and proliferation into the 3D microstructure. In-vitro fibroblast cell proliferation on elastin hydrogels
  • This example demonstrates the feasibility of fabricating GA crosslinked elastin hydrogels in an aqueous solution using high pressure CO 2 .
  • high pressure CO 2 carbon dioxide strengthened the hydrogel, whereas hydrogels that formed at atmospheric pressure were very fragile.
  • highly interconnected pores were formed with thin walled structures that resemble the natural elastin and allowed for rapid nutrient and oxygen transfer.
  • Third, the unique features of the dense gas allowed for the fabrication of channels within the 3D structure that substantially promoted fibroblast infiltration and growth throughout the matrices. Consequently, the GA crosslinked ⁇ -elastin hydrogel produced at high pressure CO 2 has high potential for in vitro applications.
  • the aim of this example was to increase the mechanical properties and also pore sizes of ⁇ -elastin hydrogel produced at high pressure CO 2 using Hexamethylene diisocyanate (HMDI) as a crosslinking agent.
  • HMDI Hexamethylene diisocyanate
  • isocyanates may react with nucleophilic functional groups such as amines, alcohols, and protonated acids.
  • Hexamethylene diisocyanate in particular was observed to react with the side chains of backbone-protected lysine, cysteine, and histidine, and to a lesser extent tyrosine, in water.
  • HMDI as a crosslinker may increase the mechanical properties of ⁇ -elastin hydrogels by enhancing the degree of crosslinking.
  • HMDI may react with other amino acids available in ⁇ -elastin structure to increase the crosslinking density and mechanical properties of fabricated hydrogels.
  • HMDI was not soluble in water; therefore, we used dimethylsulfoxide (DMSO) as a media to fabricate crosslinked hydrogel.
  • DMSO dimethylsulfoxide
  • the hydrogel was formed by the DGHF technique as described previously.
  • the hydrogels were then stored in PBS for further analysis.
  • the effects of reaction time, pressure, and crosslinker concentration on the characteristics of the hydrogel produced at high pressure CO 2 were assessed.
  • Preliminary results demonstrated that elastin was not crosslinked at reaction time below 2 hours, when 2 % (v/v) HMDI was used.
  • the crosslinker concentration increased to 5 % (v/v)
  • a hydrogel with desirable pore size and elasticity was obtained within one hour.
  • the reaction time was further increased from 1 to 2 hours, the 5 % (v/v) HMDI crosslinked hydrogel became more rigid and less elastic.
  • ⁇ is the stress (MPa)
  • F is the force (N)
  • A is the area of the specimen (mm 2 )
  • e is strain
  • ⁇ l is the change in length or compression (mm)
  • I 0 is the original length (mm) of the sample.
  • the compression modulus for the 8th cycle was obtained as the tangent slope of the stress-strain curve.
  • the energy loss based on 8 th cycle was also calculated as follows: area under loading curve - area under unloading curve
  • the average pore size of hydrogels fabricated by using 2 % (v/v) HMDI increased from 3.9 ⁇ 0.8 ⁇ m to 79.8 ⁇ 54.8 ⁇ m when the pressure was increased from 1 bar to 60 bar.
  • hydrogels fabricated by the dense gas CO 2 53 % of the pores were above 80 ⁇ m in diameter which make these hydrogels suitable for cellular growth through the 3D structures.
  • the samples produced at high pressure CO 2 were, therefore, expected to be more elastic than hydrogels produced at atmospheric conditions.
  • the compression modulus of the HMDI crosslinked hydrogel produced by high pressure CO 2 ranged from 3.99 ⁇ 0.53 KPa to 8.62 ⁇ 1.75 KPa at 40% and 80% strain, respectively ( Figure 14c).
  • the compression modulus of the hydrogels produced at high pressure CO 2 was generally lower than the one produced at atmospheric condition. This means that the samples produced at atmospheric pressure were stiffer than those produced at high pressure CO 2 . This was expected due to the presence of larger pores in the structures of the samples fabricated at high pressure CO 2 .
  • the energy loss for sample produced at high pressure CO 2 increased from 1.43 ⁇ 0.86 % to 13.16 ⁇ 1.93 %, when the stain level increased from 40 % to 80 % as shown in Figure 12D.
  • For the hydrogel produced at atmospheric pressure it increased from 4.51 ⁇ 1.54 % to 14.67 ⁇ 3.31 % when the strain level increased from 40 % to 80 %.
  • the gels formed at atmospheric pressure absorbed 4.79 ⁇ 0.15, 9.45 ⁇ 0.25, and 9.82 ⁇ 1.97 g liquid / g protein when they were swelled in PBS, water, and DMSO, respectively.
  • DMSO can destabilise the secondary structure of elastin and placticise the elastin network by increasing molecular mobility. Therefore, DMSO may plasticize the ⁇ -elastin hydrogel, resulting in an increase in segment length between crosslinks within the HMDI crosslinked gel.
  • the higher swelling ratio of the sample produced at high pressure CO 2 was due to the presence of larger pores through their structures compared to the hydrogel formed at atmospheric pressure.
  • the swelling ratio of HMDI crosslinked ⁇ -elastin using 60 bar was lower than the GA crosslinked ⁇ -elastin hydrogel fabricated at 60 bar CO 2 , which was 33.2+0.8 g H 2 O / g protein as reported in Example 4. This may due to the higher degree of crosslinking through the structures of HMDI crosslinked ⁇ -elastin hydrogel as the swelling ratio of hydrogels is correlated to the degree of crosslinking. Generally, the hydrogels with high degree of crosslinking exhibit low swelling ratio.
  • This example demonstrates the feasibility of fabricating elastin-based hydrogels with enhanced mechanical properties and pore sizes using DGHF technique and HMDI as a crosslinking agent.
  • the fabricated hydrogels had superior characteristics compared to the one fabricated using GA crosslinker.
  • HMDI crosslinked ⁇ -elastin hydrogels produced by DGHF technique had larger pores compared to GA crosslinked hydrogels due to higher solubility of CO 2 in DMSO than in an aqueous solution.
  • the fabrication of these large pores within the 3D structure substantially promoted fibroblast infiltration and growth throughout the matrices.
  • the mechanical properties was appeared to be promoted as a result of the use of HMDI.
  • the swelling ratio of the fabricated hydrogel fabricated from ⁇ -elastin/TPE mixture was in the range of 5-6 g PBS / g protein as shown in Figure 19.
  • the swelling ratio of sample exposed to high pressure CO 2 was slightly lower than the fabricated hydrogels at atmospheric condition as indicated in Figure 18.
  • Hydrogels produced at 60 bar CO 2 pressure absorbed 5 + 1.1 g liquid / g protein when they were hydrated in PBS at 37°C.
  • the gels formed at atmospheric pressure absorbed 5.9 ⁇ 1.3 g liquid / g protein when they were swelled in PBS at 37°C.
  • the lower swelling ratio of the samples produced at high pressure CO 2 may be due to the higher degree of crosslinking in their structures compared to the hydrogel formed at atmospheric pressure.
  • High pressure CO 2 facilitated the coacervation and expedited the crosslinking of TPE/ ⁇ -elastin solution.
  • the swelling behaviour of the GA crosslinked TPE/ ⁇ -elastin hydrogels produced at high pressure CO 2 was also lower than the swelling ratio of GA crosslinked ⁇ -elastin hydrogels previously reported 7 ⁇ 3.2 g PBS / g protein at 37°C.
  • the presence of TPE with high level of lysine residues in protein solution could increase the crosslinking density which resulted in a reduction in the swelling ratio of TPE/ ⁇ -elastin hydrogel.

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Abstract

L'invention porte sur des hydrogels à base d'élastine qui sont formés dans des conditions pressurisées.
PCT/AU2009/000223 2008-02-29 2009-02-27 Hydrogels dérivés de polymères biologiques WO2009105820A1 (fr)

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CN104906030A (zh) * 2014-03-14 2015-09-16 台湾生医材料股份有限公司 压力敏感性水胶及其使用方法

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Publication number Priority date Publication date Assignee Title
JP2014183886A (ja) * 2013-03-22 2014-10-02 Mie Univ 弾性組織様構造体の製造方法
CN104906030A (zh) * 2014-03-14 2015-09-16 台湾生医材料股份有限公司 压力敏感性水胶及其使用方法
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JP2015173986A (ja) * 2014-03-14 2015-10-05 タイワン バイオマテリアル カンパニー リミテッド 感圧性ヒドロゲルおよび使用方法
TWI602576B (zh) * 2014-03-14 2017-10-21 台灣生醫材料股份有限公司 壓力敏感性水膠及其使用方法

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