Gellan gum hydrogels, preparation, methods and uses thereof

Abstract

Gellan gum-based hydrogels are disclosed herein for in vitro cell culture and tissue engineering and regenerative medicine applications. Such gellan gum-based hydrogels may be used alone or combined with live cells and/or biomolecules for application in humans and/or animals. Chemical modification of gellan gum with selected ion-chelating substituents affords novel gellan gum hydrogels endowed with tunable physicochemical and biological properties. The modified gellan gum hydrogels described herein present advantages over existing hydrogel systems, including solubility, ionic crosslinking versatility, ease of formulation and injectability and greater adhesiveness within biological tissues and surfaces, whilst maintaining encapsulated cells viable during long culture periods and up-regulating the expression of healthy extracellular matrix markers.

Claims

1. A gellan gum comprising a compound having the structure of Formula I ##STR00006## wherein; R.sub.11 and R.sub.12 are independently selected from each other, R.sub.11 or R.sub.12 is hydrogen, nitro, cyano or halogen, and n is an integer from 1 to 4000.

2. The gellan gum of claim 1, wherein R.sub.11 and R.sub.12 are hydrogen and wherein n is an integer from 50 to 4000.

3. The gellan gum of claim 2, wherein n is an integer from 500 to 4000.

4. The gellan gum of claim 1, wherein the halogen group is selected from the group consisting of: fluorine, chlorine, bromine and iodine.

5. The gellan gum of claim 1, wherein said gellan gum is ionically crosslinkable by one or more monovalent, divalent or trivalent cations, or mixtures thereof, selected from the group consisting of: Na+, K+, Li+, Ca2+, Mg2+, Fe2+, Cu2+, Sr2+, Ba2+, Co2+, Mn2+, Ni2+, Sn2+, Zn2+, Fe3+, Al3+, Ga3+ and Ti3+.

6. The gellan gum of claim 1, wherein the degree of substitution of the gellan gum is between 0.1 and 30%.

7. The gellan gum of claim 1, wherein the gellan gum has a molecular weight from 100 to 2500 KDa.

8. The gellan gum of claim 1, wherein the gellan gum is in the form of a hydrogel, porous scaffold, fibres three-dimensional structure, microparticle, nanoparticle, capsule, membrane, net, gauze, disk or sprayable gel.

9. The gellan gum of claim 1, wherein the gellan gum is a medicament for a human or a non-human subject.

10. The gellan gum of claim 1, wherein the gellan gum is a treatment or therapy for bone, cartilage or soft tissue diseases or lesions.

11. The gellan gum of claim 1, wherein the gellan gum is a treatment or therapy for hyaline cartilage damage.

12. The gellan gum of claim 1, wherein the gellan gum is a treatment or therapy of a disease that is positively influenced by the chondrogenic and/or osteogenic and/or adipogenic differentiation of human mesenchymal stem cells.

13. The gellan gum pf of claim 1, wherein the gellan gum is a treatment or therapy for bone fracture, bone repair or in the treatment of osteopathies or in the treatment of osteochondritis.

14. A hydrogel comprising the gellan gum of claim 1 and a suitable solvent.

15. The hydrogel of claim 14, wherein the suitable solvent is water, a cell culture media, an aqueous saline solvent, or mixtures thereof.

16. The hydrogel of claim 14, wherein the concentration of the gellan gum is between 0.01% and 5% w/V.

17. The hydrogel of claim 14, wherein the concentration of the gellan gum is between 0.5%-2.5% w/V.

18. A composition comprising the gellan gum of claim 1, and a bioactive ingredient selected from the group consisting of a cell, a stem cell, a protein, a biomolecule, a small molecule active substance, a therapeutic agent, a diagnostic marker, and mixtures thereof.

19. The composition of claim 18, wherein the cell or stem cell are selected from a group consisting of: mammalian chondrocytes, mammalian mesenchymal stromal/stem cells, mammalian bone marrow mesenchymal stem cells, and mixtures thereof.

20. The composition of claim 18, further comprising mammalian cells and a physiological ionic solution comprising cations in an effective amount for ionic crosslinking.

21. A composition comprising: the gellan gum of claim 1; and bone marrow aspirate concentrate, growth factors, antibiotics, or mixtures thereof.

22. A composition comprising: a matrix containing the gellan gum of claim 1 or the hydrogel of claim 14; and human adipose mesenchymal stromal/stem cells.

23. The composition of claim 18, wherein the cell is an autologous cell encapsulated within the gellan gum.

24. The composition of claim 18, wherein the therapeutic agent is selected from the group consisting of: α-adrenergic agonists; β-adrenergic agonists; α-adrenergic blockers; β-adrenergic blockers; alcohol deterrents; aldose reductase inhibitors; aldosterone antagonists; amino acids; anabolics; analgesics; anesthetics; anorexics, antacids; anthelmimetics; anti-acne agents; anti-allergics; anti-androgens; anti-anginal agents; anti-anxiety agents; anti-arrythmics; anti-asthmatics; antibacterial agents and antibiotics; anti-alopecia and antibaldness agents; anti-amebics; antibodies; anticholinergic drugs; anticoagulants and blood thinners; anticolitis drugs; anticonvulsants and anti-epileptic drugs; anticystitis drugs; antidepressants; antidiabetic agents; antidiarrheal agents; antidiruetics; antidotes; anti-emetics; anti-estrogens; antiflatulents; antifungal agents; antigens; antiglaucoma agents; antihistaminics; antihyperactives; antihyperthyroid agents; antihyperlipoproteinemetics; antihypertensives; antihypotensives; anti-infectives; anti-inflammatory agents; antimalarials; antimigraine agents; antineoplastics; anti-obesity agents; antiparkinsonian agents; antidyskinetics; antipneumonia agents; antiprotozoal agents; antipruritics; antipsoriatics; antipsychotics; antipyretics; antiheumatics; antisecretory agents; antishock medications; antispasmodics; antithrombotics; antitumour agents; antitussives; anti-ulceratives; antiviral agents; anxiolytics; bactericidins; bone densifiers; bronchodilators; calcium channel blockers; carbonic anhydrase inhibitors; cardiotonics and heart stimulants; chemotherapeutics; choloretics; cholinergics; chronic fatigue syndrome medications; CNS stimulants and depressants; coagulants; contraceptives; cystic fibrosis medications; decongestants; diuretics; dopamine receptor agonists and antagonists; enzymes, estrogens; expectorants; gastric hyperactivity medications; glucocorticoids; hemostatics; HMG CoA reductase inhibitors; hormones; hypnotics; immunomodulators; immunosuppressants; laxatives; medicaments for oral and periodontal diseases; miotics; monoamine oxidase inhibitors; mucolytics; multiple sclerosis medications; muscle relaxants; mydriatics; narcotic antagonists; NMDA receptor antagonists; oligonucleotides; ophthalmic drugs, oxytocics; peptides, polypeptides; proteins; polysaccharides; progestogens; prostaglandins; protease inhibitors; respiratory stimulants; sedatives; serotonin uptake inhibitors; sex hormones; smoking cessation drugs; smooth muscle relaxants and stimulants; steroids; thrombolytics; tranquilizers; urinary acidifiers; urinary incontinence medications; vasodilators; vasoprotectants; skin protectants and sunscreens, or mixtures thereof.

25. The composition of claim 21, wherein the growth factor is TGF-β1, bone morphogenetic protein-2 (BMP-2), BMP-7 (osteogenic protein-1 [OP-1]) or cartilage-derived morphogenetic proteins CDMP-1 and CDMP-2, platelet lysates, or mixtures thereof.

26. The composition of claim 18, wherein the composition is in an injectable formulation.

27. A mesh, disk, scaffold, three-dimensional structure, strip, net, gause or membrane comprising the gellan gum of claim 1 or the hydrogel of claim 14, or the composition of claim 18.

28. A transdermal therapeutic patch comprising the gellan gum of claim 1, or the hydrogel of claim 14, or the composition of claim 18.

29. A kit for use in use in tissue engineering, regenerative medicine, or in vitro cell culture comprising a matrix containing the gellan gum of claim 1, or the hydrogel of claim 14, or the composition of claim 18, and mammalian cells.

30. A process for obtaining the gellan gum of claim 1, comprising: reacting a gellan gum according to Formula II ##STR00007## wherein n is an integer from 1-4000, M.sup.+ is a monovalent alkali metal ion chosen from the group Na.sup.+, K.sup.+ and Li.sup.+; or a group NX.sub.4 wherein X is hydrogen or a C1-C4 alkyl group chosen from methyl, ethyl, propyl or butyl, wherein the gellan gum of Formula II is reacted with, a primary amine or a secondary amine having the formula HN—R.sub.7R.sub.8, wherein R.sub.7, R.sub.8 are independently selected from each other and wherein R.sub.8 is hydrogen or a C.sub.1-C.sub.6 alkyl group, and wherein R.sub.7 represents a group according to the following formula: -(CH.sub.2).sub.g-B wherein g is an integer from 0 to 18, B is a phenol or catechol group optionally substituted on the aromatic ring by one or more, or combinations of the following groups: C.sub.1-C.sub.6 alkyl groups, hydroxyl, nitro, cyano, trifluoromethyl or halogen groups; lower alkoxy groups —OR.sub.1 wherein R.sub.1 represents a C.sub.1-C.sub.6 alkyl group; a —C(O)-R.sub.2 group, wherein R.sub.2 represents hydrogen or a C.sub.1-C.sub.6 alkyl group; a —C(O)—OR.sub.3 group, wherein R.sub.3 represents hydrogen or a C.sub.1-C.sub.6 alkyl group; a —C(O)NR.sub.4R.sub.5 group, wherein R.sub.4 and R.sub.5 represent hydrogen or C.sub.1-C.sub.6 alkyl groups; or a —SO.sub.2R.sub.6 group, wherein R.sub.6 represents hydrogen or a C.sub.1-C.sub.6 alkyl group, or B is a 3-hydroxy-4-pyridinone or a 5-hydroxypyrimidone-4(3H)-one group, and wherein the gellan gum of Formula II is reacted in the presence of at least one coupling agent and optionally in the presence of a non-nucleophilic base.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of invention.

(2) FIG. 1 represents the .sup.1H NMR spectrum of purified gellan gum (D.sub.2O, 1% w/V, 70° C.).

(3) FIG. 2 represents the .sup.1H NMR spectrum of purified gellan gum modified with 3 equivalents of dopamine (GG-DOPA3, D.sub.2O, 1% w/V, 70° C.).

(4) FIG. 3 represents the .sup.1H NMR spectrum of 6-nitrodopamine hemisulphate (NITRODOPA, DMSO-d6, 1% w/V, 70° C.).

(5) FIG. 4 represents the .sup.1H NMR spectrum of purified gellan gum modified with 1 equivalents of 6-nitrodopamine (GG-NITRODOPA1, D.sub.2O, 1% w/V, 70° C.).

(6) FIG. 5 represents the viscosity profile as a function of increasing shear rate of a solution of GG-DOPA3 in water at a concentration of 1% w/V.

(7) FIG. 6 represents a typical porous dehydrated acellular scaffold (left), rehydrated acellular scaffold (middle), and hydrogel (right) prepared from the modified gellan gums.

(8) FIG. 7 represents a typical hydrogel prepared from a modified gellan gum using whole blood as crosslinker.

(9) FIG. 8 represents the swelling characteristics of modified gellan gum scaffolds immersed in aqueous solution over time.

(10) FIG. 9 represents the water-uptake capacity of modified gellan gum scaffolds immersed in aqueous solution over time.

(11) FIG. 10a represents the fixation of GG-based hydrogels within ex vivo chondral lesions of 8 mm diameter and 2 mm thick, after testing conditions. Note complete fixation of GGp-DOPA1 and GGp-DOPA3 hydrogels and only partial fixation of GGp and GG-MA hydrogels.

(12) FIG. 10b represents a porcine joint (center image) with the six defects shown as 1 and 3-7 filled with GGp-DOPA3 hydrogels after passing all the adhesive tests in sequence. The defect shown as X is the empty control lesion.

(13) FIG. 11 represents the live/dead imaging of cells encapsulated in GG-based formulations after 21 days of culture. Green=Live; Red=Dead. Magnification=50×.

(14) FIG. 12 represents the gene expression of human adipose stromal/stem cells differentiated into chondrogenic lineage within GGp-DOPA1, GGp-DOPA3, GGp or GG-MA hydrogels. Results presented as expression ratio of cells after 21 days of culture (d21), normalized to pre-culture values (d0).

DETAILED DESCRIPTION

(15) The present disclosure provides modified gellan gums containing ion-chelating phenolic and catecholic substituents appropriate to confer improved gelation and formulation characteristics at room and physiological temperature (37° C.), and which form minimally coloured, remarkably surface-adhesive hydrogels which maintain higher cell viability for longer times after encapsulation within the hydrogel and promote up-regulation of the expression of healthy extracellular matrix markers.

(16) One skilled in the art would understand the following description, as well as terminology used herein, as to best describe the disclosed subject matter, and embodiments chosen to do so are not intended to be exhaustive or to limit the invention to the form disclosed. Alternative approaches, equivalents and conditions will be obvious to those skilled in the art.

(17) Purification of Commercial Gellan Gum

(18) Commercial gellan gum (Sigma, 5 g) was dissolved in distilled water (500 mL) with heating to 60° C. Amberlyst IR-120 (H.sup.+ form) ion exchange resin was added during 30 minutes until the solution pH stabilized at approximately 2.4. The solution was allowed to stir at 60° C. for ten minutes then filtered to remove the resin. To the filtrate was added aqueous sodium hydroxide solution until pH reached 8.5. The solution was poured onto ethanol, forming a precipitate. After stirring at room temperature for one hour, the liquid phase was decanted off and the remaining precipitate was filtered, dissolved in distilled water and dialysed against distilled water. After freezing and freeze-drying, the purified gellan gum was obtained as a white solid, 3.2 g. The .sup.1H NMR spectrum of the purified gellan gum product (FIG. 1) is in agreement with the expected structure.

(19) Modification of Purified Gellan Gum with Three Equivalents of Dopamine (GGp-DOPA3)

(20) 100 mg of purified gellan gum was dissolved in 10 mL of distilled water at room temperature. Then 0.124 g of DTMMCI were dissolved in 2 mL of water and added dropwise to the solution, which was left to stir for 30 minutes at room temperature. Next, three molar equivalents of dopamine hydrochloride (DOPA, 85 mg) was dissolved in 2 mL of water and added dropwise to the solution and the solution was then left to stir for 24 hours. Next day, the solution was removed from stirring and 10 mL of ethanol were added and the solution was left to rest for 1 hour. Then, the ethanol was decanted and the remaining precipitate was dissolved in distilled water and dialysed against distilled water for 5 days, then frozen at −20° C. and freeze-dried to give the product (GGp-DOPA3) as a white solid 128 mg. The .sup.1H NMR spectrum of the product (FIG. 2) is in agreement with the expected structure of gellan gum modified by dopamine. The degree of substitution in this case was determined as approximately 4%.

(21) By appropriately decreasing or increasing the molar equivalents of activating agent and dopamine hydrochloride used in the above example relative to the purified gellan gum starting material, it is possible to obtain modified gellan gums with degrees of substitution in the range 0.01-30%. For example, applying the procedure described above with either 1 or 7.5 molar equivalents, it is possible to obtain DOPA-modified gellan gum with lower (GGp-DOPA1) and higher (GGp-DOPA7.5) degrees of substitution (2.1 and 5.6%), respectively.

(22) Typically the DOPA-modified gellan gums contain approximately 7-10% residual moisture in the form of water. Tests for residual solvents, metals, heavy metals and leachable impurities return analytical levels normally below the detection limit of the test method.

(23) By further varying the molecular weight of the purified gellan gum starting material, it is possible to obtain DOPA-modified gellan gums with various molecular weights (Table II), as determined by gel-permeation or size-exclusion chromatography (GPC-SEC), a standard technique for determining average molecular weight (Mw), average molecular number (Mn) and intrinsic viscosity (IV) of polymeric materials.

(24) TABLE-US-00002 TABLE II Molecular weight of representative batches of GGp-DOPA3 using purified gellan gum starting material with different molecular weight. Batch Mw (KDa) Mn (KDa) Mw/Mn IV (cm.sup.3/g) #1 2080 1170 1.8 3190 #2 1454 673 2.2 2020 #3 2604 1687 1.5 4247

(25) As will be obvious to those skilled in the art, by application of the same synthetic procedure and employing appropriately substituted alcohols (R7-OH) and primary/secondary amines (HN—R7R8) instead of dopamine, the full range of modified gellan gums disclosed herein may be prepared.

(26) Nitration of Dopamine Hydrochloride

(27) To a stirred solution of dopamine hydrochloride (1.92 g, 10 mmol) in distilled water (25 mL) was added sodium nitrite (1.52 g, 22 mmol) and the solution was cooled in an ice-water bath. Thereupon, a solution of sulphuric acid (1 mL) in distilled water (10 mL) was added dropwise causing the reaction mixture to turn deep orange-red in appearance. Towards the end of addition, a thick yellow-orange precipitate formed. The mixture was then allowed to stir at room temperature overnight, then filtered and washed sequentially with ice-cold water, absolute ethanol and then diethyl ether. After drying under vacuum, 6-nitrodopamine hemisulphate was obtained as a yellow solid. The .sup.1H NMR spectrum of the product (FIG. 3) is in agreement with the expected structure.

(28) Modification of Purified Gellan Gum with One Equivalent of 6-nitrodopamine (GGp-NITRODOPA1)

(29) 500 mg of purified gellan gum was dissolved in distilled water (50 mL) at room temperature. Then 0.20 g of DTMMCI were dissolved in distilled water (5 mL) and added dropwise to the solution which was left to stir for 30 minutes at room temperature. Next, one molar equivalent of 6-nitrodopamine hemisulphate (NITRODOPA, 220 mg) was dissolved in N,N-dimethylacetamide (8 mL) and added dropwise to the solution which was then left to stir for 24 hours. Next day, the solution was poured onto 500 mL of ethanol and the precipitate was left stirring for 1 hour. Then, the precipitate was filtered off, washed with ethanol, and then dissolved in distilled water (200 mL). The product was dialysed against distilled water for 5 days, then frozen and freeze-dried to give the product (GGp-NITRODOPA1) as an orange-yellowish solid. The .sup.1H NMR spectrum (FIG. 4) of the product is in agreement with the expected structure of gellan gum modified by nitrodopamine.

(30) By appropriately decreasing or increasing the molar equivalents of activating agent and nitrodopamine hemisulphate used in the above example, it is possible to obtain modified gellan gums with degrees of substitution in the range 0.01-30%. For example, applying the procedure described above with either 3 molar equivalents, it is possible to obtain NITRODOPA-modified gellan gum with higher (GGp-NITRODOPA3) degrees of substitution.

(31) 2-Chlorodopamine hydrochloride (McCarthy, 1986) and 1-(2′-aminoethyl)-2-methyl-3-hydroxy-4-pyridinone dihydrochloride (Dobbin, 1993) were prepared as described. By application of the procedure of the above described example with these materials, the corresponding modified gellan gum hydrogel precursors were also obtained.

(32) In a preferred embodiment, the modified gellan gums, herein exemplified by GG-DOPA3, may be dissolved and maintained in deionized water, sterile or otherwise, at temperatures between 5 and 40° C., preferably between 15 and 37° C., preferably under mild agitation in less than one hour. Also in another preferred embodiment, the dissolution media may comprise cell culture media, phosphate buffer saline solution or sodium chloride saline solution.

(33) In one embodiment, the dissolution time is between 0.5 and 1 hour, preferably in less than thirty minutes.

(34) Preferred concentrations of the modified gellan gum solutions are between 0.01% and 5% w/V, more preferably between 0.1 and 4% w/V and even more preferably between 0.5 and 3% w/V.

(35) In a preferred embodiment, modified gellan gum solutions are shear-thinning liquids which (FIG. 5) that are sufficiently mobile and have suitable viscosity as to be injected by syringe. Preferred intrinsic viscosities for modified gellan gum solutions are in the range of 1000-5000 cm.sup.3/g, more preferably 1500-4500 cm.sup.3/g at a concentration of 5 mg/mL (Table II). Modified gellan gum solutions are considerably free of entrapped air and are transparent and colourless, practically colourless or lowly coloured in appearance. Modified gellan gum hydrogels formed by ionic crosslinking of such solutions are transparent and significantly free of entrapped air bubbles.

(36) Modified Gellan Gum Hydrogel Formation

(37) Initial target polymer concentration is 1.25% w/V. Weigh 12.5 mg of GGp-DOPA3 into a plastic vial and add a magnetic stirring bar. Add 1 mL of sterile water for injection. Manually and gently, make sure that the water in the vial contacts all the material for moistening. Begin magnetic stirring and continue until the material is completely dissolved at room temperature. The final solution is transparent and viscous. The formation of bubbles is minimised using ideal magnet dimensions and magnetic stirring rate indicated. If required, rest the solution to allow entrapped air bubbles to escape.

(38) After dissolution is complete, stop magnetic stirring. Add 250 μL of phosphate buffered saline (PBS with Ca.sup.2+/Mg.sup.2+) to the polymer solution (8:2 ratio of hydrogel:PBS with Ca.sup.2+/Mg.sup.2+). The divalent cations in the PBS with Ca.sup.2+/Mg.sup.2+ promote ionic crosslinking of the polymer. Final polymer concentration is 1% w/V. Gently swirl the solution for one minute. Meanwhile, in well plates, place a cylindrical plastic mold in the bottom of each well. Carefully fill the molds with the desired volume of crosslinked polymer solution without disturbing the mold. After 5 minutes, cover the mold with PBS (with Ca.sup.2+/Mg.sup.2+). Let the well plate rest for an appropriate time, then remove the molds. Leave the gels resting in the PBS solution (in suspension) for an appropriate time. Remove the gels from the well plates (FIG. 6, right).

(39) In another preferred embodiment, cell culture medium can be used instead of PBS to promote ionic-crosslinking.

(40) In a further preferred embodiment, cations other than Ca.sup.2+ and Mg.sup.2+ can be added to form modified gellan gum hydrogels via ionic cross-linking. One or more divalent and trivalent ions, or mixtures thereof, such as Fe.sup.2+, Cu.sup.2+, Sr.sup.2+, Ba.sup.2+, Co.sup.2+, Mn.sup.2+, Ni.sup.2+, Sn.sup.2+, Zn.sup.2+, Fe.sup.3+, Al.sup.3+, Ga.sup.3+ and Ti.sup.3+ may be added to the preparation in pharmaceutically acceptable salt form, such as chlorides and sulphates and hydrates thereof, at physiologically relevant concentrations. Due to the different valency and ionic radii of these ions, modified gellan gum hydrogels with a wide range of physiochemical and mechanical properties can be obtained.

(41) Surprisingly, in contrast to gellan gum and previously reported modifications of gellan gum, ions other than divalent and trivalent cations may be used to ionically crosslink the modified gellan gums herein disclosed at equivalent concentrations. In particular, physiologically relevant monovalent cations such as Na.sup.+, K.sup.+ and Li.sup.+ alone or mixtures thereof, are all able to promote formation of mechanically stable hydrogels. This is in contrast to unmodified purified gellan gum, which as mentioned earlier, does not form hydrogels in the presence of monovalent cations only. In a preferred embodiment, ionic crosslinking is promoted by 0.9% sodium chloride solution.

(42) This finding is particularly relevant with respect to physiological fluids. Thus, modified gellan gum hydrogels may be obtained by using human or animal whole blood or plasma as crosslinking agent (FIG. 7) since these are rich in monovalent cations as electrolytes. This aspect is of particular relevance within the context of microfracture.

(43) Furthermore, the surprising ability of the modified gellan gums disclosed herein to form stable hydrogels in the presence of monovalent cations overcomes the mechanical stability issues observed over time in vivo with gellan gum and methacrylated gum, related to the exchange of divalent and monovalent cations. Thus the modified gellan gums disclosed herein are able to maintain their mechanical properties over a longer period in vivo than both gellan gum and methacrylated gellan gum.

(44) Modified Gellan Gum Porous Acellular Scaffolds

(45) Modified gellan gum porous acellular scaffolds may be formed from the modified gellan gums by applying the following representative procedure.

(46) GGp-DOPA3 (375 mg) was dissolved in distilled water (30 mL) at room temperature under stirring to give a 1.25% w/V solution. Then, 7.5 mL of PBS containing Ca.sup.2+ and Mg.sup.2+ ions was placed at the centre of a plastic disc and immediately after, the GGp-DOPA3 solution was added to the plastic disk and vigorously mixed with the crosslinking solution by use of a spatula. The resulting mixture was then left to stand for thirty minutes to one hour. After this period, further PBS solution was added until the top of the disc was completely submersed. The disc was then allowed to rest for fifteen to thirty minutes. Polymeric cylinders were then formed by punching the hydrogel with a cylindrical punch having 12 mm diameter. The resulting polymeric cylinders were then transferred to 24 well plates (one disc in each well) and covered with PBS, and then changing the PBS every 9 hours three times. Then, after removing the excess PBS from each well, the plates were frozen at −80° C. for twenty-four hours. The plates are then removed from the freezer and lyophilized for three to five days to give the porous dehydrated GGp-DOPA3 scaffold (FIG. 6, left).

(47) Said porous scaffolds may be use used as tissue implants, tissue fillers or for scaffold augmentation for example in conjunction with microfracture.

(48) The GGp-DOPA3 scaffolds may be rehydrated using for example PBS solution (FIG. 6, middle) and used as such as acellular constructs, or may be used for in vitro seeding of various cell types including adipose or bone-marrow derived mesenchymal stromal/stem cells, thereby providing cell-laden implants for in vivo applications.

(49) Swelling Characteristics of GGp-DOPA3 Scaffolds

(50) Nine cylindrical scaffolds were measured (length and height) with a caliper and each scaffold was placed in a separate plate well. Each well was filled with PBS and, after 1 minute, the scaffolds were removed from the solution and immediately measured (length and height) with a caliper. Afterwards, the scaffolds were immediately placed again in the PBS solution, and the same procedure was repeated at time points of 30 minutes, 1 hour, 6 hours, 24 hours, 2 days, 3 days, 4 days, 5 days and 6 days. All the measurements were recorded, and the volume of the scaffolds at different time points was calculated as shown in FIG. 8.

(51) Water-Uptake Characteristics of GGp-DOPA3 Scaffolds

(52) Nine cylindrical scaffolds were weighed and each scaffold was placed in a separate plate well. Each well was filled with PBS. After 1 minute, the scaffolds were removed from the solution and the excessive solution was removed and the scaffolds were weighed. Afterwards, the scaffolds were placed again in the solution, and the same procedure was repeated at time points of 30 minutes, 1 hour, 6 hours, 24 hours, 2 days, 3 days, 4 days, 5 days and 6 days. All the weights were recorded at the different time point as shown in FIG. 9.

(53) Evaluation and Comparison of GGp-DOPA Hydrogel Adhesiveness—Fixation within a Chondral Lesion—Ex Vivo

(54) In the human knee joint, the medial femoral chondyle is the most frequent site of deep grade III and IV lesions. Human lesions treated for cartilage repair vary from 0.5-12 cm.sup.2 in size. In humans, the average articular cartilage thickness over 5 locations ranges from 2.2-2.5 mm, compared to 2-4 mm in pigs. With respect to cartilage thickness for induction of chondral lesions, the porcine model better resembles the scenario in humans. Thus, ex vivo conditions similar to those found in humans were used to test fixation of the new modified gellan gum-based hydrogels, including: lesion location, size, depth as well as physical stress (gravity and friction).

(55) Circular 8 mm diameter (0.5 cm.sup.2) chondral lesions were created in an articular porcine joint (critical size defect—50% of chondyle width). The cartilage in the lesion site was removed until the subchondral bone was reached and the depth of the lesions was approximately 2 mm. The defined hydrogel volume to perform the test was calculated for each lesion (V=πr.sup.2×height). GGp-DOPA1 (1% w/V), GGp-DOPA3 (1% w/V), GGp (1% w/V) and GG-MA (2% w/V) hydrogels were formulated as previously described and pipetted into the lesions, so that each lesion was completely filled. Physiological saline solution (0.9% w/V) was then used to induce crosslinking for 5 minutes. The following stress tests were performed;

(56) Gravity test: The prototype was slowly rotated upside-down, placing the lesion faced down. The prototype was vigorously shaken in order to confirm fixation of the hydrogel within the lesion (positive result) or if hydrogel dislocates from lesion (negative result).

(57) Friction test—The prototype joint was assembled and articular motion mimicked in order to confirm hydrogel fixation within the lesion (positive result) or if hydrogel breaks or dislocates from lesion (negative result).

(58) Fixation within physiological temperature was further mimicked by immersing the articular joint in 37° C. saline solution overnight.

(59) Both GGp-DOPA hydrogel formulations (GGp-DOPA1 and GGp-DOPA3) successfully withstood all stress test conditions, maintaining full integrity within chondral lesions of 2 mm thickness (FIG. 10a). In addition, it was possible to remove the 3D GGp-DOPA hydrogels fully intact from the lesion after fixation tests. On the contrary, only partial fixation of GGp and GG-MA formulations was observed within the same testing system, and hydrogel fragmentation was also observed.

(60) Regarding the concentration of hydrogel formulations, 1% w/V was found to be particularly suitable for GGp-DOPA1, GGp-DOPA3 and GGp with respect to powder dissolution time, solution viscosity and 3D hydrogel consistency and stability. For GG-MA however, a minimal concentration of 2% w/V was required to achieve similar properties. These results confirm significantly greater adhesiveness of gellan gum hydrogels modified with ion-chelating substituents at equal or lower concentrations than state of the art gellan gum hydrogels.

(61) In order to further confirm the adhesive properties of the gellan gum hydrogels, this fixation test was repeated with cartilage lesions created in several positions within a porcine joint, wherein variations in cartilage thickness could further challenge adhesion of the hydrogel. In all six positions (FIG. 10b), the hydrogel was found in place after the sequence of tests.

(62) The improved adhesive properties of the modified gellan gum hydrogels herein disclosed mean that cartilage lesions of greater size may be treated (>4 cm.sup.2), since unlike other hydrogels, the present modified gellan gum hydrogels are able to stick to surfaces of greater area. By means of example, currently available treatment options such as microfracture, ACI or MACI are only effective for treating smaller lesions between 1-4 cm.sup.2. Thus the modified gellan gum hydrogels offer advantages over current state of the art and standard of care treatment options.

(63) Assessment of Cell Viability within GGp-DOPA Hydrogels In Vitro

(64) Viability of human chondrogenic cells encapsulated within the gellan gum-based hydrogels was assessed by in vitro culture up to 21 days, this being the minimum timeframe needed for chondrogenesis. Cells were mixed with representative GG-based solutions at room temperature, to yield GGp-DOPA1, GGp-DOPA3 and GGp hydrogels at 1% w/V concentration, and GG-MA hydrogels at 2% w/V concentration, all formulations containing 5 million cells/mL.

(65) Two independent cultures with different cell types were performed, namely with human chondrocytes and human adipose mesenchymal stromal/stem cells (hASC). hASC were maintained undifferentiated or induced into the chondrogenic lineage, using chondrogenic growth factors. Individual 20 μL cellular hydrogels were pipetted and cell culture media was added to promote crosslinking of the hydrogels, resulting in stable and transferrable individual 3D structures.

(66) After in vitro culture, live and dead cells were microscopically observed by specific staining, whereby live cells are stained green by calcein AM, and dead cells are stained red by propidium iodide (FIG. 11).

(67) Regarding adipose stromal/stem cells, clearly increased live cell density is observed within the GGp-DOPA3 hydrogel, as compared to other formulations. This was evident for both differentiated and non-differentiated cells (FIG. 11, middle and right column). In addition, better cell distribution is observed within the GGp-DOPA3 hydrogel than within the GGp-DOPA1 hydrogel, while significantly void zones without cells were observed within the GGp and GG-MA hydrogels.

(68) As for chondrocyte culture, practically equivalent cell density and viability was observed amongst the various hydrogel formulations (FIG. 11, left column). These results confirm that GGp-DOPA hydrogels are able to maintain different cell types viable for long periods after encapsulation compared to state of the art gellan gum hydrogels and that by judicious choice of substitution degree, it is possible to optimize cell viability and distribution through the hydrogel.

(69) Evaluation of Chondrogenesis within GGp-DOPA Hydrogels In Vitro

(70) Collagen type II is the most abundant extracellular matrix component of healthy articular hyaline cartilage. On the other hand, presence of collagen type I is related with an unhealthy, fibrous cartilage tissue.

(71) Therefore, the expression of these two markers by human chondrogenic cells, encapsulated within the gellan gum-based hydrogels was assessed by in vitro culture up to 21 days. The experimental protocol was as described earlier for evaluation of cell viability.

(72) It was surprisingly found that the different hydrogel formulations had a very profound and unexpected impact on the expression of collagen type II and collagen type I. Among the four formulations tested, GGp-DOPA3 clearly induced expression of collagen type II to levels higher than collagen type I (FIG. 12). While this differentiating factor was evident, even more remarkable is the fact that expression of collagen type II was four times higher than collagen type I. In addition to increased expression of collagen II as compared to collagen I, the overall level of collagen type II up-regulation was on average thirteen times higher by GGp-DOPA3 than any of the other formulations, which also upholds its chondrogenic-friendly character.

(73) Thus it is surprisingly shown that both the nature of ion-chelating group and the degree of substitution of gellan gum by this substituent favours chondrogenesis. Considering potential therapeutic applications such as hyaline cartilage regeneration, certain modified gellan gum hydrogels containing ion-chelating groups with defined substitution degree as described herein have the potential to markedly improve efficacy of cell-based cartilage lesion therapeutics.

(74) Accordingly, the gellan gum hydrogels modified with ion-chelating substituents as described herein present significant advantages for in vitro cell culture and for tissue engineering and regenerative medicine by promoting long term cell viability and up-regulation of the expression of healthy extracellular matrix markers, whilst displaying improved adhesive behavior within bodily tissues and surfaces.

(75) The term “comprising” whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

(76) All references recited in this document are incorporated herein in their entirety by reference, as if each and every reference had been incorporated by reference individually.

(77) Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims.

(78) Where singular forms of elements or features are used in the specification of the claims, the plural form is also included, and vice versa, if not specifically excluded. For example, the term “a cell” or “the cell” also includes the plural forms “cells” or “the cells,” and vice versa. In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

(79) Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

(80) Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

(81) In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

(82) The above described embodiments are combinable.

(83) The following claims further set out particular embodiments of the disclosure.

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