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WO2025163341A1 - Échafaudage de gélatine réticulé, thermostable, insoluble dans l'eau - Google Patents

Échafaudage de gélatine réticulé, thermostable, insoluble dans l'eau

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Publication number
WO2025163341A1
WO2025163341A1 PCT/HU2025/050005 HU2025050005W WO2025163341A1 WO 2025163341 A1 WO2025163341 A1 WO 2025163341A1 HU 2025050005 W HU2025050005 W HU 2025050005W WO 2025163341 A1 WO2025163341 A1 WO 2025163341A1
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WO
WIPO (PCT)
Prior art keywords
gelatin
crosslinked
crosslinker
gelatine
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/HU2025/050005
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English (en)
Inventor
István HORNYÁK
Viktória VARGA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Semmelweis Egyetem
Original Assignee
Semmelweis Egyetem
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Filing date
Publication date
Application filed by Semmelweis Egyetem filed Critical Semmelweis Egyetem
Publication of WO2025163341A1 publication Critical patent/WO2025163341A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H1/00—Macromolecular products derived from proteins
    • C08H1/06—Macromolecular products derived from proteins derived from horn, hoofs, hair, skin or leather
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
    • C08L89/04—Products derived from waste materials, e.g. horn, hoof or hair
    • C08L89/06—Products derived from waste materials, e.g. horn, hoof or hair derived from leather or skin, e.g. gelatin

Definitions

  • the invention relates to the field of gel matrices and scaffold development; in particular for use in the field of tissue engineering and regenerative medicine (TERM) and provides for a water insoluble, thermostable, crosslinked gelatin matrix.
  • TAM tissue engineering and regenerative medicine
  • the fabricated matrices of the invention can be applied directly into or onto the defect and the regenerative processes are allowed to take place according to the natural healing steps.
  • scaffolds are one of the main aspects of tissue engineering and regenerative medicine (TERM), generally the intended use of scaffolds is to provide a viable environment for the growth of cells and tissues [Kim, Y. S. et al., 2018] (1).
  • the appearance of the scaffolds can be liquid, gel-like or solid, but in all cases, it is required to be compatible with the extracellular matrix (ECM) and have to be suitable to be used in a three-dimensional application [Chen, G et al. 2002] (2).
  • the fabricated matrices in TERM are generally applied directly into or onto the defect and the regenerative processes are allowed to take place according to the natural healing steps [Hinsenkamp, A., et al., 2022a] (3).
  • the degradation profile is also an important characteristic, as our aim is to develop a solid matrix that is implanted for a longer period of time, our material shall not dissolve in water and shall not melt under physiological conditions, especially pH and temperature [Bello, A.B. et al. 2020] (6).
  • the scaffolds need to enable the viability of cells, and preferably the cells can grow, proliferate and migrate in the inner structure in order to allow differentiation and remodeling in the long-term.
  • These materials can be produced using a variety of techniques, including phase separation, rapid prototyping, leaching, electrospinning, freeze drying, centrifugal casting.
  • the contents of the scaffolds are generally polymers, which can be synthetic or natural, the most important aspects from our viewpoint are biocompatibility, biodegradability, and the scaffold needs to preserve its mechanical integrity and has to promote cell attachment and viability, and to generally allow the natural regenerative functions as part of the ECM and soft tissue milieu [Chan, B.R et al., 2008] (8).
  • gelatin A material that closely resembles soft tissue is gelatin, which is derived from collagen.
  • Collagen-based biomaterial scaffolds as well as functional requirements and types of materials are reviewed by O’Brien, EJ et al. [O’Brien, F.J., 2011] (7).
  • Collagen is the most abundant protein in the human body, with nearly 1/3 of the whole protein content, and the starting material for our experiments was a partially degraded collagen: gelatin [Gelatine Handbook 2007] (9).
  • Gelatin is a biodegradable polymer that has the protein content of 85-92%, this material is non-toxic, does not induce immunological reactions, and is generally suitable for cell adhesion and viability.
  • Gelatin has been used for over a millennium both in the food, cosmetic and pharmaceutical industry, due to its versatile applicability. Due to the partial hydrolysis of collagen, gelatin is a thermoreversible material that is soluble in water at physiological temperature and can form solid gels under approximately 35°C. This low melting point is the main disadvantage of utilizing gelatin as a scaffold, as it would melt instead of slow and steady degradation [Gelatine Handbook 2007] (9). In order to improve mechanical properties, gelatin can be blended to form composites [Bikuna-Izagirre, M. et al., 2022] (10).
  • gelatin hydrogels can be further modified with the modification of the functional groups or with crosslinking the used crosslinkers include for example glutaraldehyde, genipin, formaldehyde, l-(3-dimethylaminopropyl)-3-ethyl-carbodimide hydrochloride [Tseng, H.J. et al., 2013] G-Th
  • the functional groups that can be utilized for crosslinking are generally hydroxyl, carboxyl, amino, or thiol groups [Jayachandran, B. et al., 2022] (13).
  • Gelatin a protein comprising a variety of amino acids itself, is a scaffold having all these functional groups [Gelatine Handbook 2007] (9).
  • BDDE butanediol diglycidyl ether
  • a general object of the present invention was to overcome the thermoreversibility of gelatin with the use of crosslinking to investigate a potential soft tissue material that may be used as a medical device for implantation.
  • poly(ethylene glycol) building blocks this type of crosslinker is called poly(ethylene glycol) diglycidyl ether (PEGDE).
  • PEGDE poly(ethylene glycol) diglycidyl ether
  • DVS di vinyl sulfone
  • Dias and coworkers used gelatin electrospun nanofibrous meshes, in situ crosslinked with BDDE at different concentrations (2, 4 and 6 wt%) [Dias J. R. et al., 2017] (19).
  • Crosslinking of electrospun gelatin fibers was carried out through the incorporation of BDDE in the gelatin solution immediately before fiber electrospinning to avoid the loss of configuration and provide appropriate mechanical properties that is induced through a crosslinking bath after fiber production.
  • the physicochemical and biological properties of BDDE-crosslinked electrospun gelatin meshes show no toxicity towards fibroblasts, stimulating their adhesion, proliferation and synthesis of new extracellular matrix, thereby indicating the potential of this method for skin tissue engineering.
  • the US2013157956A1 US patent application relates to a biocompatible crosslinked gelatin hydrogel composition for tissue augmentation.
  • the gel is obtained by crosslinking a 10% HA solution in 1% sodium hydroxide, using BDDE (0.5 mnol BDDE/gHA). The gel is allowed to crosslink for 24 hours at room temperature. After neutralizing with 1 M hydrochloric acid, the gel is diluted with saline sodium phosphate buffer (2.6%, pH 7.4) and dialyzed against saline sodium phosphate buffer for 48 hours.
  • a modified gelatin which is a hydroxylated gelatin and/or an aminated gelatin, was used as a starting material and reacted with an agent comprising an actinically crosslinkable group; the composites prepared can ultimately be used to produce three-dimensional engineered biological constructs. More specifically, a carboxyl group present in gelatin is first converted to a hydroxyl or amino group to produce a hydroxylated gelatin and/or aminated gelatin, and then this gelatin is reacted with an agent containing an actinically crosslinkable group to form a covalent bond.
  • the hydrogels formulations were prepared for bioprinting.
  • the invention presented in WO2016175358A1 international patent application relates to a composition for treating chronic wounds, a method for preparing the same, and a dressing material for treating chronic wounds using the same.
  • Hydrochloric acid or sulfuric acid was added to distilled water to adjust the pH to 4.0, and then collagen (1 mg/g, i.e. 0.1% which is a very low value) was added.
  • Sodium hydroxide was mixed to adjust the pH to 7.0-8.0.
  • hyaluronic acid (9 mg/g) was added to the solution.
  • BDDE as a crosslinking agent was added at a concentration of 5 mg/g (relative to solid content) in the mixed solution.
  • the crosslinking step was performed at 30 °C for 12 hours.
  • Epidermal growth factor and fibroblast growth factor were added to the crosslinked matrix, and the final product was prepared by injecting the gel into a mold and freeze-drying.
  • an aqueous coating fluid comprising gelatin at a concentration of at least 1 wt% and gelatin hardener at a level from 1-200 effective pmole hardener per gram of coating fluid.
  • the gelatin is prepared from the hydrolysis of ossein using sodium or potassium hydroxide; these materials are not used here in the crosslinking step.
  • the KR101866678B1 Korean patent protects an invention, designed to improve the efficiency of the crosslinking reaction between hyaluronic acid hydrogel and the crosslinking agent and to use an aqueous alkali solution at the crosslinking step.
  • hyaluronic acid was dissolved in NaOH and then a crosslinking agent, BDDE was added and a hydrogel was formed.
  • gelatin is mentioned as an option, no example is provided and no amine functional group is mentioned.
  • the chemical crosslinking agent comprising epoxide can be BDDE, ethylene glycol diglycidyl ether (EGDE), polyethylene glycol diglycidyl ether (PEGDE), glycerol polyglycidyl ether, and diglycerol polyglycidyl ether.
  • EGDE ethylene glycol diglycidyl ether
  • PEGDE polyethylene glycol diglycidyl ether
  • glycerol polyglycidyl ether glycerol polyglycidyl ether
  • diglycerol polyglycidyl ether diglycerol polyglycidyl ether
  • CN105801920A discloses a biomedical material containing a chemical crosslinking composition, which may include, but is not limited to, a cellulose derivative, a gelatin and at least one crosslinking agent, wherein the cellulose derivative and gelatin are interposed between the cellulose derivative and the gelatin by the at least one crosslinking agent.
  • the crosslinking reaction took place in an oven at 40 ° C and dried to form a crosslinked film.
  • a pH 5-8 is applied throughout the process set in a buffer solution with strong acid or base.
  • CN103992499A discloses a 3D uniform porous scaffold material in order prepare an artificial bone scaffold material having a three-dimensional structure, prepared by the following steps
  • Step 1 Dissolving collagen in sodium carbonate solution to achieve a final mass fraction of collagen of 10-30%;
  • Step 2 Adding nano-hydroxyapatite, which seems to be an essential component, to the mixture of step one;
  • Step 3 Dispensing the mixture obtained in the second step into a mold
  • Step 4 The mold is frozen at -80 ° C for 3-6 h, and then vacuum dried (lyophilized) for 48 h;
  • Step 5 The first lyophilized product of the fourth step is cross-linked at 37-50°C in the cross-linking agent solution, wherein the pH of the cross-linking system is 8-11, for 24-36 h;
  • Step 6 Subjecting the sample after cross-linking in step 5 to secondary crosslinking reaction wherein the pH of the cross-linking system is 3-6., the reaction temperature is 37-50 ° C, the reaction time is 24-36 h; Step 7 : Washing the sample;
  • Step 8 Vacuum-drying the sample.
  • the first crosslinker may be a diglycidyl ether.
  • the result is a hard, bone-like material.
  • the experiments are performed in the solid phase, at room temperature, without the use of buffers, and at a high pH at which the material would otherwise decompose within 4 hours.
  • the prior art crosslinked gelatin products prepared in solution are formed as insoluble precipitates which have no specific form and are to be obtained or recovered from the solution. Any form for the product can be provided thereafter only.
  • the reaction mixture usually has to be heated to elevate the temperature above the melting temperature of gelatin.
  • a lyophilized, solid gelatine matrix or scaffold is wetted or soaked with an alkaline crosslinking solution to arrive at a formed product.
  • the lyophilized gelatine scaffold has typically a shape and thereby also the crosslinked gelatine product maintains this shape which is essential unchanged by crosslinking, i.e. the product is solid as well.
  • the crosslinker solution is added under mild or cool temperatures, e.g. room temperature or below.
  • the invention relates to the following embodiments.
  • cross-linkable gelatin matrix comprising primary amine-groups, wherein preferably cross-linkable gelatin matrix is provided in a form (shape or mould),
  • said lyophilized gelatin matrix is permeated by an aqueous crosslinker solution comprising
  • alkali hydroxide preferably 0.2 M to 1 M alkali hydroxide (preferably 0,5% to 10%, more preferably 0.8% to 4.0 % alkali hydroxide, preferably NaOH; preferably at least (or above) pH 11, preferably at least (or above) pH 12 or 13), preferably 0.2% (W/V) (0.05M) to 20% (5M) NaOH,
  • water soluble crosslinker agent capable of crosslinking primary amine groups of gelatine, preferably said water soluble crosslinker agent provided in 0.1 to 30 % (V/V), (per volume of the crosslinker solution),
  • the lyophilized gelatin matrix comprises at least 70% gelatine.
  • the lyophilized gelatin matrix is prepared from gelatine only.
  • the lyophilized gelatin matrix comprises at most 30% non-gelatine gel-forming agent.
  • the non-gelatine gel-forming agent is hyaluronic acid.
  • the alkali hydroxide is applied in 0.2 M to 1 M concentration (0.8% to 4.0 % NaOH) with 5-15% (V/V) cross-linker agent.
  • the crosslinker is a crosslinker capable of acting on amine groups (amine- crosslinker).
  • the crosslinker is an epoxy (polyepoxy) crosslinker.
  • the epoxy crosslinker is a diglycidyl crosslinker e.g. as defined below e.g. in paragraph 5 or 6.
  • the epoxy crosslinking agent is a divinyl crosslinker as defined below e.g. in paragraph 7.
  • the gelatine matrix is prepared from a gelatin solution containing 1 to 10% gelatine.
  • the method of the invention is a method for the preparation of a soft tissue implant comprising the crosslinked gelatin hydrogel prepared according to the invention, wherein form of the crosslinked gelatin hydrogel is adapted to an implantation site in an animal.
  • the animal is a vertebrate animal, in particular an amphibian, a reptile, a fish, a bird or a mammal, preferably a bird or a mammal, highly preferably a mammal.
  • the animal is poultry.
  • the animal is a mammal.
  • the animal a companion animal.
  • the animal is livestock.
  • the invention also relates to a method for the preparation of a crosslinked gelatin hydrogel preferably according to paragraph 1 , wherein
  • gelatine solution is provided in a mould (or to have a shape),
  • the gelatin solution optionally provided in a form (or having a shape), is lyophilized to prepare a solid, lyophilized gelatin matrix, until the water content of the matrix is below 10 w/w%,
  • the solid lyophilized gelatin matrix is permeated by the crosslinker solution (or soaked into a crosslinker solution), preferably without a buffering agent, said solution comprising
  • the lyophilized gelatin matrix comprises at least 70%, preferably at least 80%, in particular at least 90% gelatine.
  • the lyophilized gelatin matrix essentially consists of gelatine as a gel-forming agent.
  • the lyophilized gelatin matrix comprises at most 30% non-gelatine gel-forming agent.
  • the non-gelatine gel-forming agent is hyaluronic acid or nothing.
  • the solid lyophilized gelatin is allowed to cross-link, preferably at a 0 to 15 °C temperature, for 30-60 hours to form a crosslinked gelatin hydrogel.
  • the cross-linking temperature is preferably 0 to 10°C or 4 to 10°C.
  • the alkali hydroxide is applied in 0.2 M to 1 M concentration (0.8% to 4.0% NaOH) with 5-15% (V/V) cross-linker agent.
  • the invention relates to a method for the preparation of a crosslinked gelatin hydrogel according to the invention, wherein
  • gelatin solution containing 1 to 10% gelatine, more preferably from 2 to 8% gelatine is provided, preferably said gelatin solution is prepared between 35 and 60 °C, more preferably between 45 and 55 °C, until a homogenous solution is formed,
  • the gelatin solution is lyophilized to prepare a solid, lyophilized gelatin matrix, wherein the gelatin solution is frozen, preferably frozen at -40 to -120 °C preferably at -60 to -100 °C, more preferably -70 to -90 °C, in particular at about -80°C and freeze dried afterwards with the collector temperature is between -40 to — 60 degrees, preferably between -45 and -55 °C and at the reduced pressure of between 0.5 and 50 Pa, preferably between 1 and 10 Pa, until the water content of the matrix is below 10 w/w%, preferably below 5 w/w%
  • the solid lyophilized gelatin matrix is soaked into a crosslinker solution comprising 0,05M to 5M alkali hydroxide, preferably 0.2% (W/V) (0,05M) to 20% (5M) NaOH, more preferably 0.5 to 10% NaOH, preferably without a buffering agent, and water soluble epoxy crosslinker of 0.1 to 30% (V/V), preferably 0.5 to 20 % (V/V), more preferably 1 to 10% (V/V) (or alternatively % of the weight of the original solution), wherein preferably said NaOH solution is freshly prepared,
  • crosslinked gelatin hydrogel preferably with water, preferably from 1 to 5 times, preferably 2 to 4 times, or until the unreacted crosslinkers are removed from the solution.
  • the epoxy crosslinker is a water soluble epoxy, preferably a diglycidyl crosslinker.
  • the gelatin solution is lyophilized to prepare a solid, lyophilized gelatin matrix, wherein the gelatin solution is frozen and freeze dried afterwards,
  • the gelatin solution contains 1 to 10% gelatine or 2 to 8% gelatine
  • the gelatin solution is lyophilized to prepare a solid, lyophilized gelatin scaffold the water content of the which is below 5 w/w%,
  • the solid lyophilized gelatin matrix is soaked into a crosslinker solution comprising 0.2-1 M alkaline hydroxide solution, preferably 0,5 to 10% NaOH, or 0.8% (W/V) (0,2M) to 4% (IM) NaOH, without a buffering agent, and water-soluble epoxy crosslinker of 0.5 to 20 % (V/V), more preferably 1 to 10 % (V/V), wherein preferably said alkaline hydroxide, preferably NaOH solution is freshly prepared,
  • the solid lyophilized gelatin is allowed to cross-link, preferably at a temperature of 0 to 15 °C or 0 to 10°C, for 30-60 hours to form a crosslinked gelatin hydrogel,
  • the gelatin solution contains 2 to 8% gelatine is provided, wherein said gelatin solution is prepared between 35 and 60 °C, more preferably between 45 and 55 °C, until a homogenous solution is formed, the gelatin solution is lyophilized to prepare a solid, lyophilized gelatin matrix, preferably at -70 to -90 °C, and preferably freeze dried afterwards with the collector temperature between -45 and -55 °C and at the reduced pressure of between 1 and 50 Pa, until the water content of the matrix is below
  • the solid lyophilized gelatin matrix is soaked into a crosslinker solution comprising 0,5 to 10% NaOH without a buffering agent, and water-soluble epoxy crosslinker of 1 to 10 % (V/V).
  • crosslinker capable of crosslinking primary amino groups is selected from the group consisting of water soluble polyepoxy compounds, preferably ethylene glycol diglycidyl ether (EGDE), or polyethylene glycol diglycidyl ether (PEGDE), butanediol diglycidyl ether (BDDE), particularly preferably PEGDE and BDDE.
  • EGDE ethylene glycol diglycidyl ether
  • PEGDE polyethylene glycol diglycidyl ether
  • BDDE butanediol diglycidyl ether
  • the water-soluble epoxy crosslinker has the general formula (I) wherein R1 is C4-C50, preferably C4-C20, or preferably C4-C10 polyether moiety (having at least two ether oxygens), preferably R1 is a C4-C20, or preferably C4-C10 polyether moiety having the general formula (2) wherein R2 is selected from the group consisting of a C2-C18 alkyl, a C2-C18 alkene, a C2-C18 alkyne, (preferably a C2-C8 alkyl, more preferably a C2-C4 alkyl), C2-C18 alkylether, a C2-C18 alkene-ether, a C2-C18 alkyne-ether (preferably a C2-C8, more preferably a C2-C4 alkylether), preferably R2 is selected from the group consisting of butyl, isopropyl, ethy
  • R3 is selected from ethyl, methyl and H, preferably methyl and H.
  • crosslinker is a compound having formula (IV), wherein n is 1 to 12, preferably 1 to 4, more preferably 1 to 2.
  • the gelatin solution contains 2 to 8% gelatin,
  • the gelatin solution is lyophilized to reach a water content lower than 5 w/w% of the matrix
  • the crosslinker solution comprises 0.5 to 10% NaOH, without a buffering agent, and the water-soluble epoxy crosslinker of 0.5 to 20 % (V/V), - the cross-linking is carried out at a temperature of 0 to 15 °C, preferably 0 to 10°C, for 8-72 hours, preferably 30 - 60 hours to form a crosslinked gelatin hydrogel,
  • the crosslinked gelatin hydrogel is washed, preferably with water.
  • crosslinked gelatin hydrogel is a crosslinked gelatin hydrogel scaffold preferably for implantation.
  • the temperature is 0 to 30°C for 30 - 60 hours to form a crosslinked gelatin hydrogel, and the reaction time is at least 8 hours, preferably 8 to 72 hours.
  • crosslinker is as defined in any of claims 5 to 6. wherein n is 1 to 4, more preferably 1 to 2.
  • the gelatin solution is provided by 3D printing.
  • Providing in a form means that the gelatine solution to be lyophilized has a shape.
  • the gelatine solution can have a shape due to e.g. moulding, casting into a form, or cutting (forming) into a shape, or by alternative methods e.g. 3D printing.
  • a soft tissue implant comprising the crosslinked gelatin hydrogel product obtained (or obtainable) by crosslinking a freeze-dried gelatine matrix by permeating said freeze-dried gelatine matrix by an aqueous crosslinker solution via the gelatine amine groups, wherein said gelatine hydrogel product is porous and resistant heat sterilization, preferably resistant to heat at least up to 130 °C, preferably at least up to 150°C.
  • said gelatin hydrogel product maintains the porosity of the freeze-dried gelatine matrix but cross-linked.
  • said crosslinked gelatin hydrogel product has a solid porous material.
  • the crosslinked freeze-dried gelatine matrix is obtained by a method according to any of paragraphs 1 to 9.
  • the crosslinked gelatin hydrogel product is capable of withstanding heat sterilization.
  • the crosslinked gelatin hydrogel product is a solid porous material, preferably forming a solid porous body.
  • a soft tissue implant comprising the crosslinked gelatin hydrogel product or a crosslinked gelatin hydrogel product obtainable according to a method of paragraph 1 to 9, wherein the compression strength of the crosslinked gelatin hydrogel product is not lower than that of 80% of the starting non-crosslinked gelatine, preferably not lower than that of the crosslinked gelatin hydrogel product, and/or wherein the tensile strength of the crosslinked gelatin hydrogel product is not lower than that of 80% of the starting non-crosslinked gelatine, preferably not lower than that of the crosslinked gelatin hydrogel product.
  • both the compression strength and the tensile strength are similar, i.e. within ⁇ 50%, preferably within ⁇ 30%, more preferably within ⁇ 20% to those of native gelatine, preferably to those of a gelatine from which the preparation starts from.
  • the piece of crosslinked gelatin hydrogel product is flat shaped, i.e. its two dimensions (e.g. length and width) are significantly longer than the third dimension (thickness).
  • the piece of crosslinked gelatin hydrogel product is block shaped.
  • the piece of crosslinked gelatin hydrogel product has a shape provided by a mould or form as defined in paragraph 11.
  • the piece of crosslinked gelatin hydrogel product can be cut into an appropriate size.
  • crosslinked gelatin hydrogel product is obtained by a method of any of paragraphs 5 to 6.
  • R3 is selected from ethyl, methyl and H, preferably methyl and H, and
  • X is a gelatine chain linked via an amine bond
  • Q is selected from the group of a gelatine chain and a hyaluronic acid chain.
  • the crosslinked gelatin hydrogel product is a soft tissue implant.
  • the gelatin solution is provided by 3D printing.
  • the lyophilized gelatin scaffold is provided in a form by 3D printing of the gelatin.
  • the crosslinked gelatin hydrogel product is supplied with cells, preferably serves as a scaffold for cells.
  • said piece having at most 100 ml.
  • said piece (or portion) of crosslinked gelatin hydrogel product has a shape.
  • said piece (or portion) of crosslinked gelatin hydrogel product is a solid porous body.
  • a soft tissue implant comprising the crosslinked gelatin hydrogel product according to any of paragraphs 9 to 15 or 16, and having a shape adapted to an implantation site of a patient.
  • the soft tissue implant comprises a piece of a crosslinked gelatin hydrogel product according to the invention.
  • the soft tissue implant comprises multiple pieces of crosslinked gelatin hydrogel product according to the invention and is adapted to the form of the implantation site.
  • the soft tissue implant according to paragraph 18, said implant comprising a plurality of scaffolds arranged in a multi-layer stacked configuration.
  • said patient is treated by surgery and said crosslinked gelatin hydrogel is used as a filler.
  • said patient is treated by a correction surgery after an injury.
  • said patient is injured in a soft tissue and having a malformation or a soft tissue damage.
  • said patient has a developmental disorder wherein a tissue is damaged or a tissue part is missing.
  • said crosslinked gelatin hydrogel is implanted into or adjacent to an extracellular matrix.
  • a lyophilized cross-linkable gelatin matrix in the manufacture of a soft tissue implant comprising a crosslinked gelatin hydrogel as defined in any of claims 10 to 15, for use in the treatment of a patient as defined in paragraph 18, in particular of a patient in need of a soft tissue implant.
  • said patient is treated by surgery and said crosslinked gelatin hydrogel is used as a filler.
  • the patient is a vertebrate patient.
  • the vertebrate patient is an amphibian, a reptile, a bird, a fish or a mammal.
  • the patient is a mammalian, highly preferably a human patient.
  • a method for use of the soft tissue implant for implantation for treatment of a tissue defect and injury comprising steps: administering said implant into the site of injury in the patient.
  • the patient is a vertebrate patient.
  • the vertebrate patient is an amphibian, a reptile, a bird, a fish or a mammal.
  • the patient is a mammalian, highly preferably a human patient.
  • the invention relates to a method of surgery of a patient in need of soft tissue replacement or augmentation, wherein a soft tissue implant comprising a crosslinked gelatin hydrogel as defined in any of claims 9 to 15, preferably 10 to 15 is provided, the site of implantation is prepared in said patient by surgical means, wherein a soft tissue implant comprising a crosslinked gelatin hydrogel as defined in any of claims 9 to 15, preferably 10 to 15 is provided, the site of implantation is prepared in said patient by surgical means, wherein a
  • said patient is treated by a correction surgery after an injury.
  • said patient is injured in a soft tissue and having a malformation or a soft tissue damage.
  • said patient has a developmental disorder wherein a tissue is damaged or a tissue part is missing.
  • said crosslinked gelatin hydrogel is implanted into or adjacent to an extracellular matrix.
  • the soft tissue implant is a medical device.
  • the implant is a face implant, an implant for soft tissue injury, e.g. fat tissue injury of muscle tissue injury.
  • the implant is used in the field of tissue engineering and regenerative medicine (TERM).
  • EGDE ethylene glycol diglycidyl ether
  • PEGDE polyethylene glycol diglycidyl ether
  • - x means the quantity (in mg) of gelatin is used when preparing the scaffold
  • - y means the quantity (in mg) of hyaluronic acid is used when preparing the scaffold
  • - HA means hyaluronic acid
  • - N if present, is the type of the cross-linker, e.g. B means BDDE, P means PEGDE,
  • scaffold 50G-12.5HA the scaffold was prepared 50 mg gelatin and 12.5 mg hyaluronic acid.
  • gelatin refers to a polypeptide polymer comprising helical poly-amino acid fibers including any gelatin, whether extracted by traditional methods or recombinant or biosynthetic in origin, or to any molecule having at least one structural and/or functional characteristic of gelatin, preferably one or more characteristic selected from amino acid composition, secondary and/or tertiary protein structure, mechanical properties like tensile strength, gel strength, and spectral properties like FT-IR spectra, NMR spectra, and also molecular weight.
  • gelatin is built up from amino acids wherein the majority of the amino acids forming gelatine are selected from valine, leucine, isoleucine and aspartic acid.
  • Polypeptides from which gelatin can be derived are polypeptides such as collagens, procollagens, and other polypeptides having at least one structural and/or functional characteristic of collagen.
  • a polypeptide could include a single collagen chain, or a collagen homotrimer or heterotrimer, or any fragments, derivatives, oligomers, polymers, or subunits thereof, containing at least one collagenous domain (Gly-X-Y region).
  • the gelatin is a “recombinant gelatin” or a “recombinant gelatin-like protein” which terms specifically contemplate engineered sequences not found in nature, such as altered processed collagen sequences, e.g.
  • sequences that is altered, through deletions, additions, substitutions, or other changes, from a naturally occurring collagen sequence or which are prepared by recombinants synthesis from collagen encoding genes.
  • sequences may be obtained from, for example, suitable altered collagen polynucleotide constructs.
  • gelatin as used in reference to the present invention encompasses both a gelatin material comprising gelatin polypeptides, as well as an individual gelatin polypeptide.
  • Crosslinker refers to a reactive chemical compound that is able to introduce covalent intra- and inter-molecular bridges in organic molecules.
  • Preferred crosslinking agents useful in the present invention are bifunctional protein crosslinkers which contain two or more reactive groups which covalently attach via a spacer to functional groups on proteins, preferably on protein amine groups, i.e. which are capable of crosslinking amine groups, preferably primary or secondary amine groups in alkaline conditions.
  • Preferred crosslinking agents useful in the present invention are diglycidyl or epoxy crosslinkers, which are particularly preferred, as well as divinyl crosslinkers.
  • crosslinkers used herein are homobifunctional crosslinkers or heterobifunctional crosslinkers, preferably homobifunctional crosslinkers.
  • crosslinked gelatin refers to gelatin that has been derivatized by reaction with, e.g., one or more small chemical moieties such as diglycidyl or epoxy crosslinker.
  • An epoxy-crosslinked gelatin (or gelatin hydrogel) contains gelatin chains crosslinked with a crosslinker comprising at least one epoxy bond, preferably a diglycidyl crosslinker, preferably resulting in a covalent bond formed on the primer amino groups of the gelatin chains.
  • gelatin solution is a solution wherein gelatin is the solute and the solvent is an aqueous solvent, wherein the solution may be in a liquid form (sol) or in a solid (gel) form.
  • a “hydrogel” as used herein is a colloid network of polymer chains that are hydrophilic and contain or is capable of containing water; preferably the network is formed from the polymer chains by crosslinking.
  • the polymer is gelatin.
  • a matrix is a bulk of homogenous solid material, preferably gel material.
  • a scaffold as used herein is a matrix wherein a cross-linking reaction has occurred.
  • a crosslinked gelatine hydrogel is a material which is useful for treatment of a patient.
  • a soft tissue implant is a medical device having a shape or form comprising a crosslinked gelatine hydrogel and is adapted for implantation into an implantation site of a patient.
  • An alkylether group is an ether wherein both side of the oxygen is an alkyl or an alkylene group.
  • administration includes introducing or applying any implant, like prosthesis or replacement, made of the hydrogel of the invention into a living body, e.g., animal body, human body.
  • Such administration can be topical i.e., may be applied to a particular place on or in the body.
  • the hydrogel of the invention is administered inside the subject’s body it can be performed by invasive surgery, preferably by minimally invasive methods.
  • Preferred administration is grafting or implanting.
  • subject refers to vertebrate animal, e.g., a reptile, an amphibian, a fish, a bird or a mammal; preferably a warm-blooded mammalian, particularly a human being.
  • the hydrogel of the invention is to be administered to a subject.
  • the use of the hydrogel and thus an implant made of the hydrogel or comprising the hydrogel, may be e.g. medical or cosmetic.
  • patient includes a subject that receives or is considered to receive either therapeutic treatment to restore health or improve a disease condition and prophylactic treatment including maintaining health or improving a healthy condition.
  • treatment is thus meant to include both prophylactic and therapeutic treatment, in particular to treat, replace, repair or augment a tissue at a target site.
  • Figure 1 The appearance of the crosslinked matrices.
  • the matrices were 5 mm in diameter and were 2 mm thick.
  • the appearance of the matrices was generally sponge-like
  • Figure 2 Sponge-like structure of the scaffold under microscope (Zeiss AXIO Imager. A 1)(A): lOx and (B): 20x magnification.
  • Figure. 3. Surface of the scaffold using BDDE as crosslinker.
  • Figure 4. Surface of the scaffold using DVS as crosslinker. Small cracks are visible on the surface of the matrix, indicated with white arrows.
  • Figure 5 Surface of the scaffold using PEGDE as crosslinker.
  • A 50x
  • B lOOx
  • C 500x
  • D lOOOx
  • E 2000x
  • F 5000x magnification.
  • Figure 6 Number of pores and average relative area of the pores using the three different crosslinkers. The calculation was done using ImageJ using the 50x magnification images.
  • Figure 7. Microscopic images of the explanted scaffold after 1 month.
  • A shows the actual size of the implant which was ⁇ 5mm as seen on scale.
  • B highlights the integration of vascular structures (indicated with red arrows).
  • Figure 8. Newly formed blood vessels after 1 month (indicated with red arrows). 2x (A), 4x (B), and lOx (C) magnification.
  • Figure 9 Micrographs of HA-GEL after autoclaving. From left to right, top to bottom: 125GEL, 100GEL25HA, 75GEL50HA, 50GEL5HA, 25GEL100HA, 125HA.
  • Figure 14 FTIR spectra of native and crosslinked gelatin. GEL is visible in the bottom of the figure, and from bottom to top, the 1, 3, and 5 V/V % BDDE (Fig. 5A) and 1, 3, and 5 V/V % PEGDE (Fig. 5B) crosslinker containing matrices are visible.
  • Figure 16 Load - Deflection curves of native GEL scaffolds and 5 V/V % BDDE crosslinked GEL (50G5B). Both native GEL samples and crosslinked samples were tested in triplicates.
  • Figure 17. 17a: Average maximum tensile load of native GEL scaffolds and 1, 3 and 5 V/V % BDDE crosslinked GEL. Both native GEL samples and crosslinked samples were tested in triplicates. 17b: Cumulated diagram on the tensile strength test measurement results with gelatine based hydrogels and with HA and GEL composite hydrogels.
  • Figure 18. Toxicity and attachment of the 5 V/V % BDDE scaffolds for MSCs - it is shown that cells were able to attach to the surface of the scaffolds after 24 hours and were able to proliferate after 168 hours.
  • 18A sample prepared from 50 mg gelatin with 5% BDDE
  • 18B sample prepared from 50 mg gelatin and 12.5 mg hyaluronic acid
  • Figure 19A Optical microscopic image of a gelatin-based cell scaffold, removed from the black 6 male mouse after 3 months.
  • Figure 19B Optical microscopic image of a gelatin-hyaluronic acid-based cell scaffold, removed from the black 6 male mouse after 3 months.
  • Figure 20 The cell attachment and proliferation on the two optimal scaffolds using MSCs. The absorbances were measured after 24 and 168 hours of culturing. 5 w/w % GEL, crosslinked in 20 pL BDDE and 300 pL 1 w/w % NaOH (I.), 5% GEL, crosslinked in 40 pL BDDE and 600 pL 1 'w/'w% NaOH (IL).
  • Figure 21 Cell attachment and proliferation on scaffolds fabricated using three BDDE crosslinker concentrations (1, 3, 5 V/V %) and one 5 V/V % PEGDE crosslinker. The absorbances were measured after 24 (A) and 168 hours (B) of culturing.
  • FIG 22 Hystological images of the explants. Different tissue staining methods were used on the samples, (A) and (B): Movat’s pentachrome staining were used to demonstrate collagen and reticular fibers (yellow), nuclei and elastic fibers (black/blue), and fibrin and muscle (red); (C) and (D): Hematoxylin-eosin staining were used to localize extracelluar matrix features (pink) and cell nuclei (blue/purple). The presence of red blood cells and newly formed blood vessels is indicated with red arrows.
  • Figure 23. Elastica van Gieson (EvG) staining of the explants.
  • H&E Hematoxylin and Eosin
  • MG Masson and Goldner
  • the present inventors have worked on the construction of a gelatin-based scaffold, in particular scaffolds useful in tissue engineering and regenerative medicine (TERM).
  • the scaffold was prepared by freeze- drying and was crosslinked afterwards with epoxy (diglycidyl) crosslinkers having low toxicity but still effective enough to chemically modify gelatin.
  • epoxy diglycidyl
  • a very efficient modification could be achieved under highly alkaline conditions, e.g., with 0,05-5 M alkaline hydroxide, e.g., in 0.1 to 2%, in particular about 1% NaOH.
  • the most promising concentration for the crosslinkers were the 1, 3 and 5 V/V% BDDE and PEGDE matrices.
  • GEL non-crosslinked gelatin gel
  • BDDE diglycidyl crosslinker
  • the crosslinked weight is important to see when the added BDDE starts to increase the starting weight. It was surprising that already with the use of 1 % BDDE the crosslinked weight wasn’t significantly different from the starting weight, whereas a slight and not significant weight gain was observable with the use of 10 and 20 % BDDE and that, while a swelling ratio difference was observable, the swelling ratio didn’t decrease significantly even with the use of 10 and 20 % BDDE.
  • the compression and tensile strength were also tested, the tested mechanical parameters showed similarity to the native gelatin samples.
  • the scaffolds were water insoluble, resistant to collagenase enzyme and were able to withstand heat sterilization.
  • the matrix of the invention was also tested in vitro to see if hMSCs could adhere to and proliferate on the matrix.
  • the matrix of the invention is suitable for medicinal purposes as a soft tissue implant that can be an important tool in regenerative medicine (see Figure 9).
  • BDDE, DVS and PEGDE as potential crosslinkers were investigated to produce a biomimetic gelatin-based scaffold that does not become soluble under physiological temperature and is suitable to be heat sterilized, without further degradation.
  • DVS was unable to lead to a heat stable matrix, thus we continued with BDDE and PEGDE.
  • TRIS native GEL
  • reaction can be enhanced with the use catalysators as the prior art advises [La Gatta, A et al., 2016] (21), however, to reduce the potential toxic materials the inventors chose to avoid the use of any catalysators and to use the crosslinker and simple NaOH solution only.
  • reaction temperature initially room temperature was applied, however, later it has been found that conditions under 4°C provide an even better condition for an evenly crosslinked material.
  • the crosslinking at 4°C resulted in higher crosslinked weight and lower swelling ratio.
  • the reaction was more effective and less degradation occured under cooled conditions.
  • This effect is preferably achieved below 15°C and wherein the reaction mixture is liquid, preferably below 10°C, e.g. 0-15°C, preferably 0-10°C.
  • the reaction may be carried out e.g. at 4°C or 0-6°C or even below 0 °C, provided the reaction mixture is not frozen. Typically, it is not necessary to thermostate the mixture but may be useful.
  • the starting weight, the crosslinked weight and the swelling ratio of the scaffolds were measured. It has been found that degradation and crosslinking take place simultaneously, which implies that the crosslinked weight is important to see when the added crosslinker starts to increase the starting weight. It was surprising that with the use of 1% BDDE and PEGDE the crosslinked weight was significantly lower than the starting weight and in the case of 3 and 5 V/V % BDDE and PEGDE, significant weight gain was observable. However, the swelling ratios were not significantly different in either concentration, neither in the case of BDDE nor with PEGDE. The larger weight difference in the PEGDE crosslinked matrices, is probably due to the ethylene glycol chain that adds more weight to the crosslinked composition in the case of PEGDE compared to BDDE.
  • collagenase was used in the case of the material that was produced with 20 % BDDE as crosslinker and was compared to native 5% GEL in water and in collagenase. After significant differences were found, the present inventors carried out an enzymatic degradation screening with all the used BDDE ratios to see the effect.
  • the use of collagenases is well known in the modeling of in vivo degradation of biomaterials [Wassenaar, J.W. et al., 2016, Bailey, A.J.
  • the 1 and 3 % BDDE containing matrices showed the most degradation but the 5 % BDDE containing one was as stable as the higher BDDE containing scaffolds.
  • characterization with the 1, 3, and 5% BDDE containing ones was carried out.
  • the crosslinking via an epoxy crosslinker takes place between the carbon atom adjacent to the oxygen atom in the epoxy ring and between the primer amino group [Kircher, R. et al, 2022] (26), forming the molecules that are visible on Scheme 1 and 2 showing a general reaction between a primer amino group containing molecule and BDDE (Scheme 1) or PEGDE (Scheme 2) with the expected products (Figure 5).
  • the present invention can be used in tissue engineering and regenerative medicine (TERM).
  • the fabricated matrices in TERM are generally applied directly into or onto the defect and the regenerative processes are allowed to take place according to the natural healing steps [Hinsenkamp, A. et al., 2022 (a)] (3).
  • the present scaffold can used as a medical device, as it can fulfill the applicable quality management related standards [Hinsenkamp, A. et al., 2020] (4).
  • the materials used are typically biocompatible and can meet the requirement of safety for human implantation [Hinsenkamp, A. et al., 2022 (b)] (5).
  • the use of cells and growth factors in TERM is also a popular approach, however, in the present the inventors’ aim was that the scaffold can be used in itself, as well.
  • the freeze-drying method chosen in the present invention results in a particular advantage.
  • the crosslinked derivatives of gelatin are prepared in a solution or in a melted form, or e.g.. in the case of electrospinning, the crosslinking takes place during the fiber formation, thus it was surprising that we were able to crosslink gelatin using it in a freeze dried form.
  • the relative surface of the gelatin matrix increased with the sublimation of water, compared to the starting solid gelatin or a gelatin film.
  • the alkalic solution of the crosslinker effectively reacted with the functional groups in the gelatin chains and crosslinking was also achieved.
  • the solvents in the present invention are aqueous solutions, in particular water, high temperature is not required, thus, biomimetic materials are optimal for the preparation with the use of freeze drying.
  • the lyophilized, solid gelatin matrix or scaffold preferably having a specific form, is wetted or soaked with an alkaline crosslinking solution to arrive at a formed product.
  • the lyophilized gelatin scaffold has typically a shape and thereby also the crosslinked gelatin product maintains this shape which is essential unchanged by crosslinking, i.e. the product is solid as well.
  • the crosslinker solution is added under mild or cool temperatures, e.g. room temperature or below.
  • porosity is an inherent feature of the scaffolds of the invention.
  • lyophilization increases the specific surface of the gel.
  • a lower gelatin concentration also increases the specific surface.
  • TE tissue engineering
  • gelatin a material processed from collagen, was known as a food additive.
  • our age saw a vast variety of its applications from photographic materials to its use in regenerative medicine.
  • Gelatin is prepared by the processing of collagen either by acidic, alkaline or enzymatic method.
  • Acid treatment can be carried out by treating the collagen with a strong acid like sulfuric, hydrochloric, or phosphoric acid referred to as type A gelatin. This relatively long hydrolysis method is often used materials like porcine skin collagen.
  • Alkaline processing is typically used for more complex collagens, e.g. for bovine collagen; and may require a longer time.
  • the resulting type is the so-called alkaline gelatin (type B).
  • a third and most recent method is the enzymatic hydrolysis of collagen. It requires a shorter time that with the previous two methods and results in a less degradation of gelatin. Moreover, a high purity is more easily arrived at.
  • gelatin any type of gelatin can be used.
  • BDDE butanediol diglycidyl ether
  • Crosslinking may take place between two functional groups of the gelatin chains, which are usually the - OH or the -NH2 groups.
  • the preferred functional groups are the amino groups
  • the suitable crosslinkers are able to either form conjugated bonds on a gelatin chain or form covalent bonds between gelatin chains.
  • These crosslinker types are usually belong to the aldehydes (e.g., glutaraldehyde and formaldehyde), carbodimides, polyepoxy compounds, e.g. ethylene glycol diglycidyl ether (EGDE), or polyethylene glycol diglycidyl ether (PEGDE), butanediol diglycidyl ether (BDDE).
  • water soluble epoxy crosslinkers are applied having at least one, preferably at least two epoxy groups capable of binding to the gelatin chain, preferably to the amino groups (preferably -NH2) of gelatin, by opening up of the epoxy ring while the -O- group forms an -OH and the methylene carbon is linked to the amine nitrogen.
  • the crosslinker has two epoxy rings at its two ends which, once crosslinking has taken place, both are linked to gelatin chains.
  • An example for the crosslinking reaction chemistry is shown on Scheme 2.
  • the water-soluble epoxy crosslinker has the general formula (I) wherein R1 is a poly ether moiety (having at least two ether oxygens) as defined in the Brief Description chapter or in the appended claims or in the Examples.
  • Ri is poly ether moiety having the general formula (2), as defined in the Brief Description chapter or in the appended claims, linked at its ends to the epoxy rings as shown in formula (I).
  • R2 is selected from the group consisting of butyl, isopropyl, ethyl,
  • R1 is a polyether moiety, of a length as defined in the Brief Description chapter or in the appended claims or in the Examples, having the general formula (3) wherein n is a number as defined herein, in particular in said description sites.
  • the crosslinker is a compound having formula (IV), wherein n is a number as defined herein, in particular in said description sites.
  • Epoxy crosslinkers in preferred embodiment diglycidyl crosslinkers, are known in the art.
  • the scaffold of the invention can be characterized by the following particular features.
  • the crosslinked weight of the scaffold is higher than the starting weight when more than 1 V/V % crosslinker is added, in particular 2-30% or 2-6%, preferably 3-20 V/V % or 3-5 V/V % crosslinker is added to the scaffold and the swelling ratio gets lower as the amount of crosslinker increases.
  • the crosslinked weight is significantly higher when 3 and 5 V/V % PEGDE was used compared to 1 V/V % PEGDE, whereas the swelling ratio is less sensitive to the crosslinker ratio.
  • the scaffold of the invention is able to withstand collagenase degradation at least for 4 hours.
  • This is shown in figure 3 which compares the degradation of native gelatin in H2O, native gelatin and 20 V/V% BDDE crosslinked gelatin both in 1 mg/ml collagenase. It is clear from the presence of the primer amine group in the solution that the aqueous samples (GH) showed relatively little decomposition, whereas from 4 hours, there was significant difference between the GH and GC groups and between the GC and crosslinked GEL in collagenase solution (XGC) group, and this difference remained significant even after 48 hours.
  • the scaffold of the invention is able to withstand heat sterilization (e.g. autoclaving), i.e. is heat resistant at least up to 130°C or preferably at least up to 150°C.
  • heat sterilization e.g. autoclaving
  • the highest degradation was found in the 1 V/V% BDDE-containing matrix (50G1B), followed by the 3 V/V % BDDE containing one after 48 hours, the difference was significant. There was no significant difference between the degradation in the 5, 10, and 20 V/V% BDDE containing matrices after 48 hours
  • the free amino acid content of the scaffold of the invention is significantly reduced in comparison with native gelatin, e.g. which is the starting material for gel production.
  • the crosslinked scaffolds have a similar tensile strength compared to the starting gelatin material. Surprisingly, based on load deflection measurement studies the crosslinked samples were less rigid, i.e. softer and easier to compress and the load - compression diagram was less steep when using crosslinked samples.
  • the curves represent how each material is able to withstand compression, generally a more rigid material has a steeper curve as the compression increases, a softer material has lower steepness.
  • crosslinked scaffolds of the invention are suitable for the attachment and proliferation of cells.
  • Group II. had more alkaline solution and crosslinker during the reaction, thus it would have been expectable that the structure would degrade easier compared to group I. and III., which had less alkalic solution and crosslinker.
  • group I. was implanted for only 4 weeks, compared to group III., which was implanted for 12 weeks, we would have expected larger differences in the scaffold area, and blood vessel formation.
  • the relative area of the scaffolds did not differ significantly according to the evaluation with the use of three different staining, which lead to the conclusion that with our crosslinking method, the scaffolds did not degrade significantly compared to the size of the scaffolds at the time of the implantation.
  • Our goal was to fabricate a biocompatible scaffold that is non-toxic, non-immunogenic, does not degrade over three months.
  • the envisioned product that we aim to develop is a permanent flexible implant, that is regulated as a medical device, thus the pre-liminary results seem to support our expectations.
  • Gelatin was purchased from Gelita, hyaluronic acid from Contipro.
  • Crosslinkers BDDE and PEGDE were purchased from TCI Chemicals, DVS was purchased from abcr Gmbh, and other materials were purchased from Merck.
  • Gelatin scaffolds were prepared at the concentration of 50 mg/ml. 100 mg of gelatin was weighed on an analytical balance and dissolved in 2 ml of reverse osmosis filtered (RO) water using a ThermoShaker at 50 °C. The resulting solutions were lyophilized at -55 °C and 5 Pa for 24 hours. Gelatin samples after freeze-drying were cut into quarters with a scalpel. BDDE was mixed with 1% w/w NaOH solution, which was used to provide alkaline condition for the crosslinking reaction, and the mixture was pipetted onto the freeze-dried quarters.
  • RO reverse osmosis filtered
  • the crosslinker was used in 1% V/V, 3% V/V, 5% V/V, 10% V/V and 20% V/V with 150 pl NaOH solution.
  • a scale-up step was also included, when possible, in this case the entire 100 mg GEL containing matrix was used and was put in the freshly mixed crosslinker/NaOH, that also contained 4 times the reagents compared to the quarters described above.
  • the crosslinking reaction took place for 48 h at room temperature.
  • the crosslinked gels were washed three times with 5 ml of RO water, sterilized using heat sterilization, cut with 5 mm diameter biopsy punches and then freeze-dried under aseptic conditions to reach the form that was tested in vitro and in vivo.
  • the physical appearance of the scaffolds was examined, including the size and porosity. As it is visible on figure 1, the dry matrices retained their shape and the porous structure. The inner structure was further visualized using higher magnification which is presented in figure 2.
  • gelatin scaffolds were also prepared at the concentration of 50 mg/ml.
  • solutions with volume of 20 ml 1000 mg gelatin was weighed on an analytical balance and dissolved in 20 ml of reverse osmosis filtered (RO) water using a Thermo-Shaker at 50 °C.
  • Solutions with volume of 30 ml 1500 mg gelatin was measured and dissolved in 30 ml RO water.
  • the resulting solutions were lyophilized at -55 °C and 5 Pa for 24 hours.
  • Gelatin samples after freeze-drying were mixed with BDDE and 1% w/w NaOH solution in a Petri dish.
  • Gelatin scaffolds were prepared at the concentration of 50 mg/ml. For 1 ml solution, 50 mg gelatin was weighed on an analytical balance and dissolved in 1 ml of reverse osmosis filtered (RO) water using a Thermo-Shaker at 50 °C. The resulting solution was freeze dried at -55 °C and 5 Pa for 24 hours. The dry matrices were mixed with DVS and 1% w/w NaOH solution. 20 pl DVS and 300 pl 1% NaOH were mixed, then it was placed in an ice bath and the freeze-dried gelatin matrix was placed in the vial. The crosslinking reaction was performed for 48 h at 4 °C. The crosslinked matrices were washed with 5 ml of RO water three times and were freeze-dried again to reach the final form.
  • RO reverse osmosis filtered
  • gelatin scaffolds were prepared by dissolving 50 mg of gelatin in 1 ml H2O at 50 °C. The resulting solutions were allowed to cool then were frozen and freeze-dried. 40 pl of PEGDE was mixed with 600 pl 1% w/w NaOH solution, and the freeze-dried matrices were immediately added to the crosslinking mixture. The crosslinking reaction was carried out for 48 h at room temperature. The crosslinked gels were washed three times with 5 ml of RO water and were freeze-dried again to obtain the final shape.
  • the surface microstructures of the cells that were seeded and cultured on the scaffolds were examined by a scanning electron microscope (SEM) (JEOL JSM-6380LA). Before microscopic observation the membranes were fixed by 2.5% glutaraldehyde for 20 min. Dehydration was performed with increasing concentrations of ethanol (50%, 70%, 80%, 90%, 100%) for 5 min each. After dehydration, samples were treated with 0.5 mL of 100% hexamethyldisilazane (HMDS) for 5 min and they were left in the safety cabinet overnight to allow excess HMDS to evaporate. After drying, the samples were sputter-coated with gold (JEOL JFC-1200 Fine Coater, 12 mA, 20 s) and examined under SEM. The middle region of the surfaces were scanned using 50x, lOOx, 250x, 500x, lOOOx, 5000x, and 10 OOOx magnification. The images of the sample surfaces are visible on figures 3 to 5.
  • SEM scanning electron microscope
  • the scaffolds can support cell attachment, which indicates that these were sterile, non-toxic and had similar pore sizes.
  • the main purpose of the development was to fabricate a material that is suitable to be implanted for a longer period of time.
  • the structure of the matrices prepared with DVS appeared to be fragile.
  • PEGDE diglycydil-ether matrices which were originally prefferred because of the results of the mechanical experiments
  • PEGDE was found to have a rather uneven surface. Therefore the 5 V/V % BDDE matrices were chosen for the in vivo experiments, and were implanted for either one month or three months in the back of BL6 mice.
  • the matrices were examined using light microscopy and histology.
  • the explanted materials didn’t show any sign of infection, and generally live tissue was starting to infiltrate the matrices. Both fibrous tissue on the outside and blood vessel formation in the inside was visible, as shown in the demonstrative images figure 7 and figure 8.
  • HA-GEL samples of 125 mg each were prepared, which contained different proportions of hyaluronic acid and gelatin (see Table 1).
  • Sodium hydroxide (1 mL, 1%) and the crosslinking agent (5% DVS) were added to the mixtures, homogenized with a vortex and centrifuged (Hinsenkamp A., 2020).
  • samnle m gelatin mhyaluronic acid (mg) (mg)
  • the HA-GEL lyophilized mixtures were also examined by light microscopy to determine the differences between the gelatin concentrations.
  • Optical microscopic measurements were carried out by a Leica M80 microscope at 1.25x magnification.
  • the size of the scaffold was 5 mm diameter and 2-3 mm thickness (depending on the swelling ratio).
  • Degradation of native lyophilized * matrices from 2 ml 5 % w/w gelatin were measured at 7 different pH values for 168 hours.
  • 5 ml buffer solution was added to the native gelatin quarters and threes parallel measurements were taken. Gelatin leaching was then monitored using a Nanodrop UV-VIS spectrophotometer. The absorbance was measured at 205 and 230 nm.
  • the used crosslinkers can be harmful for the human body, thus it is important not to leave any unreacted crosslinkers in the scaffold and to use as little crosslinker amount as possible, while maintaining the required stability.
  • the swelling ratio did not further decrease above 5 V/V % crosslinker (data not shown).
  • Figure 11 shows the starting and crosslinked weight of gelatin scaffolds, including the swelling ratio after 48 hours of crosslinking, with the use of 1-3-5 V/V % BDDE (Fig. 11A) and PEGDE (Fig. 11B) in 600 pl 1% NaOH.
  • the crosslinked weight of the scaffolds was higher than the starting weight when 3 and 5 V/V % was added to the scaffold and the swelling ratio gets lower as the amount of crosslinker increases, however, the crosslinked weight was only significantly higher when 3 and 5 V/V % PEGDE was used compared to 1 V/V % PEGDE, the swelling ratios did not differ significantly from each other in either group.
  • HA-GEL combinations were appropriate if the 25 mg HA + 50 mg GEL + 5 pl BDDE sample was applied. In other cases, the samples disintegrated after washing with distilled water, which was either due to insufficient mixing or insufficient amount of crosslinker.
  • the first measurement aimed to observe and model the in vitro degradation of native and 20 N/N % BDDE crosslinked gelatin scaffolds using 5 ml 1 mg/ml collagenase enzyme (Serva, Collagenase NB 4G) in RO water.
  • 5 ml 0.2 mg/ml collagenase was added to native and crosslinked * matrices, which contained 1%, 3%, 5%, 10%, 20% BDDE.
  • the samples were allowed to react on a thermostated shaker at 300 rpm and 25 °C for 48 hours.
  • the absorbances were measured with a Nanodrop One spectrophotometer at 205 and 230 nm.
  • the stability of the native 5 % GEL in water and in collagenase solution was compared to the crosslinked 5 % GEL using 20 V/V % BDDE.
  • the expectation was that the crosslinked samples would degrade less than the native ones, and that those in water would degrade only slowly.
  • Figure 12 reports on the comparison of the degradation of native GEL in H2O (GH), native GEL in 1 mg/ml collagenase (GC) and 20 V/V % BDDE crosslinked GEL in 1 mg/ml collagenase (XGC) at 230 nm (Fig. 12).
  • GH aqueous samples
  • GC collagenase
  • XGC collagenase solution
  • Figure 13 is a plot illustrating the comparison of the degradation of crosslinked gelatin with 1, 3, 5, 10, and 20 V/V % BDDE in 0.2 mg/ml collagenase at 230 nm. As it is visible in the figure, according to our expectations, the highest degradation was in the 1 V/V % BDDE containing matrix (50G1B), followed by the 3 V/V % BDDE containing one after 48 hours, the difference was significant. There were no significant difference between the degradation in the 5, 10, and 20 V/V % BDDE containing matrices after 48 hours, but these were all significantly different from 50G1B.
  • the solution shows the presence of NAG formed from hyaluronic acid during enzymatic degradation by a color reaction, which is directly proportional to the concentration of NAG, measured by spectrophotometer at 585 nm (see Table 4). This was used to test the resistance of the combination to the action of the enzyme hyaluronidase.
  • FTIR measurement was performed to compare the spectra of the native gelatin with the crosslinked gels. Besides, we compared the different BDDE amount containing gels spectra to each other, in order to find out which is the most proper to stabilize gelatin.
  • the samples were prepared as described before [Hinsenkamp A. 2021] (14). The measurement was performed with a Bruker Vertex 80v spectrometer. It was equipped with a high sensitivity mercury-cadmium-telluride detector and a single reflection diamond ATR accessory. 128 scans were performed with a resolution of 2 cm 1 in the range of 400-4000 cm 1 .
  • the free amino acid content which is the unreacted amino acid content of the scaffolds was measured in order to decide the effectiveness of the crosslinking.
  • the free amino acid content of the scaffolds are shown in Figure 15. showing the relative absorbance of native and crosslinked gelatin matrices at 335 nm.
  • the free amino groups are expressed as relative absorbances compared to the absorbance of native freeze-dried gelatin.
  • the free primer amino groups decreased significantly in every group that contained crosslinker compared to native GEL even in the case of 1 V/V % BDDE and PEGDE. There was no significant difference between the absorbance of the negative control and the 3 V/V % or 5 V/V % crosslinker containing scaffolds’ absorbance.
  • the average maximum tensile load was measured and compared between the native and crosslinked GEL matrices. There were no significant differences between the different samples, thus the crosslinked scaffolds had similar tensile strength compared to the starting material.
  • the next step was lyophilization, which is performed overnight until the samples are completely dry. The whitish discs were then quartered and weighed individually.
  • a crosslinking agent was prepared containing 5-10 to 20 pl BDDE and 1% 150 pl NaOH per sample. The order is important, i.e., the NaOH is measured first, as the BDDE would react quickly. It is crucial that the crosslinking agent is pipetted onto the HAGEL samples immediately after suspension so that it covers the entire surface. Otherwise, the scaffolds would disintegrate without crosslinker. After 24 hours, the materials should be washed three times with 5 ml of distilled water per sample.
  • the concentration of hyaluronic acid was fixed i.e., 1.6% hyaluronic acid solution in liquid form.
  • nine, i.e., 3-3-3, composite samples were prepared by first measuring 30, 40 and 50 mg gelatin, adding the 1.6% hyaluronic acid solution to the samples, and then melting the mixtures at 50 °C. To make them more miscible and homogeneous, the samples were centrifuged at 1000 G for 8 min and allowed to homogenize for another 24 h. This centrifugation procedure had to be repeated the next day and the next step was to lyophilize the samples, which took a whole night because to make the samples completely dry.
  • the finished whitish disks were then cut into quarters and weighed individually.
  • the sample quarters were used for a BDDE crosslinking experiment, where the 150 pl l%NaOH solution was measured first. To this, 10, 20, and 30 pl BDDE was added. It is crucial that the crosslinker is pipetted onto the hyaluronic acid gelatin samples immediately after suspension so that it covers the entire surface. Otherwise, the scaffolds will disintegrate without the crosslinker. After 24 hours, the materials should be washed with 5 ml/sample of distilled water. After 24 hours, it was found that the 30 and 40 mg samples had completely disintegrated because the structure was too loose due to the lack of crosslinker.
  • FBS fetal bovine serum
  • DMEM Dulbecco Modified Eagle Medium
  • bovine fibroblast growth factor bFGF
  • PEST penicillin-streptomycin
  • the stem cell medium was sterilized using a sterile syringe filter.
  • the cells were then centrifuged at 600 G for 5 min, the supernatant was washed, and the cells were plated in fresh medium.
  • Cell culturing was performed were cultured in T75 TC treated culture flasks in an incubator at 37 °C, 5% CO2, and 95% humidity, which allowed the cells to adhere sufficiently to the surface of the flask. The medium had to be refreshed three times a week.
  • the stem cell medium was removed and washed the cells with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Accutase was used to pick up cells deposited at the bottom of the flask, as Accutase breaks the adhesion bonds between the cells and the flask surface.
  • the reaction was also stopped with stem cell medium and the mixture was centrifuged again.
  • a Biirker chamber cell count method was performed, after which the cells could be used for experiments.
  • Cytotoxicity measurement was carried out using an XTT assay.
  • the XTT assay Cell Proliferation Kit II (XTT), Roche, Mannheim, Germany) is a colorimetric assay, which is applied to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. The assay detects surviving cells after toxic exposure, and it can also be tested in vivo and in vitro.
  • the colorimetric assay is based on the fact that the mitochondrial enzymes of healthy cells reduce a yellow XTT dye (i.e., 2,3- Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) into an orange compound called formazan.
  • a yellow XTT dye i.e., 2,3- Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide
  • the fading orange color indicates a decrease in the number of viable cells proportional to the increase in toxicant dose.
  • An increase in the number of viable cells results in an increase in the total activity of mitochondrial dehydrogenases in the sample.
  • the amount of orange formazan formed is directly related to this increase, which is monitored by spectrophotometrically, that is, via measuring absorbance.
  • An intermediate electron acceptor (EC) is used to measure sensitivity. This facilitates the reduction of XTT as it is able to withdraw electrons from the cell surface (Desai, Sharav et al., 2011).
  • the hydrogels were placed on a 96 well micrometer plate and 200 pl of stem cell medium was pipetted in. The medium was refreshed every 2 days. 50 pl of EC, XTT mixture was added to each well and incubated at 37 °C for 4 hours. On the next day the viability of the seeded cells was measured using Cell Proliferation Kit II (XTT; Roche, Mannheim, Germany), according to the manufacturer’s instructions, on half of the membrane-containing wells to measure the number of attached cells. The rest of the membranes with the seeded cells were cultured for 6 more days The absorbance of the soluble formazan produced by the viable cells was then measured at 460 nm using an ELISA plate reader. The reference wavelength used was 650 nm. XTT measurements were performed after 24 hours and on the 7th days and the results were compared. The measured absorbance of the scaffolds at 460 nm are shown in Fig. 20.
  • the gelatin-based scaffolds containing hyaluronic acid were tested on black6 male mice, approximately 80-90 days old. The implantation was performed in the same way with the optimal 50G 12.5HA 30/200 gel as the pure gelatin base. These scaffolds were implanted in the back of mice, subcutaneously for 3 months. After implantation, the same method was followed, the implant was removed from the mouse after termination, and it was observed that after 3 months, the process of vascularization was successfully initiated in this case, as well ( Figure 19B.).
  • mice 3-4-month-old C57B1/6N mice were used. Before anesthesia, the weight of the mice was measured, which ranged between 30-40 grams. Anesthesia was induced with 3% isoflurane, and after placing the mice on a heating pad, the concentration was reduced to 2.5% for the duration of the procedure. The dorsal area of the mice was depilated using hair removal cream at the designated site, followed by cleaning with alcohol. A skin incision of approximately 1 cm was made on the back, and subcutaneous pockets were carefully created along the incision line using fine scissors. The implants were inserted into the prepared pockets, and the incision was closed with 5/0 silk sutures.
  • mice received antibiotics (Amoxicillin / clavulanic acid, Antapharma 1000 mg / 200 mg - 0.05 * mouse weight * 100 pl) intraperitoneally to prevent inflammation and infection. The day after surgery, the mice were returned to their original bedding cages.
  • antibiotics Amoxicillin / clavulanic acid, Antapharma 1000 mg / 200 mg - 0.05 * mouse weight * 100 pl
  • the animals in were sacrificed by cervical dislocation, and their backs were depilated. An incision was made to expose the implants, which were observed by a light microscope (Leica M80; Leica Microsystems, Wetzlar, Germany) and then removed. The procedure was repeated after twelve weeks with the 20 animals in the 12-week group. After the scaffolds were removed, they were fixed in 4% formaldehyde, their weights were measured, and they were sent for histology measurements.
  • tissue samples were dehydrated in a graded series of alcohol and embedded in polymethylmethacrylate.
  • Slices in the longitudinal direction of the implant were cut with a laser microtome (TissueSurgeon, LLS ROWIAK GmbH, Hannover, Germany) and stained with Hematoxylin & Eosin (HE), Elastica van Gieson (EvG), Masson and Goldner (MG), and Movat’s Pentachrome (MP) .
  • Slice thickness was 30 pm. Scanning and digitalizing for evaluation were performed using an optical microscope Zeiss AXIO Imager. Al (Carl Zeiss MicroImaging GmbH, Gottingen, Germany) at 2x, 4x, lOx or 20x magnification. Samples were evaluated qualitatively in terms of structure and degradation of the scaffold (preserved fiber structure), reaction of surrounding tissue (cell infiltration), and cell migration into scaffolds.
  • Figures 7 and 8 depict the explanted scaffolds 1 month after the implantation. On both of these images newly formed blood vessels can be seen.
  • red blood cells was similar in all the samples groups as well as the area of the scaffold and the cytoplasm, there were no significant differences in the values in either group.
  • the present inventors worked on the construction of the scaffold, starting with the reaction parameters and optimal amount of reagents.
  • the scaffold was prepared by freeze-drying and was crosslinked afterwards, the crosslinkers were chosen to be well known and to have low toxicity but still be effective enough to chemically modify gelatin.
  • the chemical modification was assessed with FTIR and the compression and tensile strength was also tested, the tested mechanical parameters showed similarity to the native gelatin samples and supported that the product is useful in regenerative medicine.
  • the scaffolds were water insoluble, resistant to collagenase enzyme and were able to withstand heat sterilization, rendering it stable under in vitro and in vivo circumstances.
  • the matrix of the invention can be used to adhere cells and protein factors, e.g. hMSCs and proliferate on the matrix we hope to be able to ultimately use it for medicinal purposes as a soft tissue implant that can be an important tool in regenerative medicine.
  • compositions of crosslinked gelatin-based scaffolds were tested in vitro and in vivo. Three different crosslinkers were used in vitro and the optimal composition was chosen for in vivo testing.
  • the surfaces of the scaffolds were observed with SEM and in the case of di vinyl sulfone (DVS): small cracks appeared and the structure was rigid.
  • DVS di vinyl sulfone
  • PEGDE poly(ethylene glycol) diglycydil ether
  • a preferred scaffold contained 5 V/V % butanediol diglycidyl ether (BDDE) was tested for both one month or three months in the back of BL6 mice.
  • the explants were assessed using analytical techniques, including microscopic imaging and histological analysis and it was found that cells, connective tissue, and extracellular matrix (ECM) were all able to successfully infiltrate the scaffolds and did not induce any inflammation.
  • the implants seem to promote blood vessel formation, support the adherence of adipose tissue as confirmed by optical microscopy and histological evaluations.

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Abstract

L'invention concerne le domaine des matrices de gel et du développement d'échafaudages ; en particulier pour une utilisation dans le domaine de l'ingénierie tissulaire et de la médecine régénérative (TERM) et concerne une matrice de gélatine réticulée, thermostable, insoluble dans l'eau. Les matrices fabriquées de l'invention peuvent être appliquées directement dans ou sur le défaut et les processus régénératifs peuvent avoir lieu suivant les étapes de cicatrisation naturelle.
PCT/HU2025/050005 2024-01-29 2025-01-29 Échafaudage de gélatine réticulé, thermostable, insoluble dans l'eau Pending WO2025163341A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080181964A1 (en) * 2003-12-04 2008-07-31 Kyekyoon Kim Microparticles
CN103333508A (zh) * 2013-06-28 2013-10-02 陕西巨子生物技术有限公司 一种注射用胶原蛋白水凝胶及其制备方法
CN103992499A (zh) * 2014-04-21 2014-08-20 陕西巨子生物技术有限公司 一种3d均匀多孔支架材料及其制备方法
CN105801920A (zh) * 2014-12-24 2016-07-27 财团法人工业技术研究院 化学交联组合物、含其的生物医学材料以及其用途

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US20080181964A1 (en) * 2003-12-04 2008-07-31 Kyekyoon Kim Microparticles
CN103333508A (zh) * 2013-06-28 2013-10-02 陕西巨子生物技术有限公司 一种注射用胶原蛋白水凝胶及其制备方法
CN103992499A (zh) * 2014-04-21 2014-08-20 陕西巨子生物技术有限公司 一种3d均匀多孔支架材料及其制备方法
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