Water insoluble, thermo-stable, crosslinked gelatin scaffold

Abstract

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, thermo- stable, crosslinked gelatin matrix. 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.

Description

[0001] WATER INSOLUBLE, THERMO STABLE, CROSSLINKED GELATIN SCAFFOLD

[0002] FIELD OF THE INVENTION

[0003] 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. 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.

[0004] BACKGROUND ART

[0005] The use of scaffolds is 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).

[0006] 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).

[0007] In order to enable a scaffold development to ultimately be regulated as a medical device, it has to fulfill the applicable quality management related standards [Hinsenkamp, A., et al., 2020] (4), one of these requirements is to use materials, which were found to be safe for human implantation [Hinsenkamp, A., et al., 2022a] (5). Another important requirement for the application as a soft tissue is to have a pore size distribution that is suitable for cells to grow, the material shall have a relatively high surface, and shall be non-toxic, biodegradable and the mechanical properties shall closely resemble soft tissue. 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). Besides the mechanical properties, 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).

[0008] 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). However, 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).

[0009] Safety of butanediol diglycidyl ether (BDDE) has been a question, however, according to previous works of the present inventors it has been found that the use of BDDE is safe if the product is handled correctly and non-reacted crosslinker is removed; moreover, the biocompatibility and cell viability did not change with the use of this material [Hinsenkamp, A. et al., 2021 and Hinsenkamp, A. et al., 2022 (c)] (14, 15). BDDE was also used to crosslink fish derived gelatin, and the mechanical properties were examined, however, not as a biologically applicable material [Czerner, M. et al., 2020] (16).

[0010] 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.

[0011] Zeeman R. et al. discovered earlier that BDDE is suitable to crosslink amino groups and was used to modify collagen from sheep [Zeeman, R et al., 1999, Zeeman, R et al., 2000] (17) (22), however, in their work both the starting material and the end product was water insoluble, and the reaction took place in an aqueous solution with the use of buffers.

[0012] Additionally, another family of epoxy crosslinkers contains poly(ethylene glycol) building blocks, this type of crosslinker is called poly(ethylene glycol) diglycidyl ether (PEGDE). The present inventors have also tested this material as a potential crosslinker, as well as di vinyl sulfone (DVS) which was effective in experiments when hyaluronic acid was the starting material [Hinsenkamp, A. et al., 2021] (14).

[0013] Vargas et al. have crosslinked type-B gelatin with EGDE (from 3 to 15 wt.%) between pH of 7.4 to 11, buffered with boric acid and citric acid. [Vargas, G., 2008 18]. They found that at pH higher than 9, “the swelling ratio increased, due to the denaturation in the gelatin as well as a negative interference from an excessive amount of OH“ ions in the crosslinking reaction” (page 3658), however, they used 15 w / w % gelatin, and buffers in liquid phase at elevated temperature (37 °C). The best crosslinking reaction between gelatin and EGDE was achieved at pH=9 and EGDE concentration of 10 wt.%. The reaction occurred at 37 °C, in solution within 90 minutes.

[0014] In their work, 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 authors in their next publication [Dias J. R. et al., 2018.] modified the method and developed hybrid structures, including individual polycaprolactone and gelatin gel electrospun meshes. The gel preparation method was the same. The healthcare or medical application of crosslinked gelatin is known in the patent literature, for example, the CN111195373A Chinese patent application relates to gel particles prepared from crosslinked gel particles and gel liquid - with a mass ratio of 3:7 to 6:4 - wherein the size of the crosslinked gel particles are 1 to 20 pm and formed by grinding from the polymerized gel. The crosslinking reaction starting from 15-30 w / w% gelatin in aqueous solution by carbodiimide and N-hydroxy succinimide in the presence of genipin took 16 hours on pH 5.5-7.8, while the temperature is relatively low, 4-10 °C.

[0015] 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.

[0016] In the W 02011088213 Al international patent application, preparation of hydrogels and their medical applications are presented. 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.

[0017] 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. Then, 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.

[0018] According to the invention described in the US patent US6589326B1 , an aqueous coating fluid is presented 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.

[0019] 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. As an example, hyaluronic acid was dissolved in NaOH and then a crosslinking agent, BDDE was added and a hydrogel was formed. Although 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. The procedure is very long (5 to 30 days) and no idea of crosslinking a lyophilized gelatin support is raised.

[0020] Sung, Hsing-Wen et al. [Sung, Hsing- Wen et al., 1996] analyze the crosslinking characteristics of epoxyfixed porcine tendon in terms of effect of pH, temperature and fixative concentration. The authors have observed that the denaturation temperature and fixation index of the epoxy-fixed tendons increased rapidly at pH 10.5. The authors also note that it is known that extremes of pH may cause denaturation of proteins by disrupting weak attractive forces.

[0021] In US2008 / 0181964AI uniform crosslinked gelatin microparticle is prepared from droplets.

[0022] 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.

[0023] 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

[0024] Step 1 : Dissolving collagen in sodium carbonate solution to achieve a final mass fraction of collagen of 10-30%;

[0025] Step 2: Adding nano-hydroxyapatite, which seems to be an essential component, to the mixture of step one;

[0026] Step 3: Dispensing the mixture obtained in the second step into a mold;

[0027] 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;

[0028] 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;

[0029] Step 8: Vacuum-drying the sample.

[0030] The first crosslinker may be a diglycidyl ether. The result is a hard, bone-like material.

[0031] There is still a need for improved soft tissue implants, or for improved gelatin gel scaffolds that can be commercialized by themselves, e.g., as medical devices; are, however, biologically tolerable (or biologically compatible) and suitable for use in TERM, among others together with cells and growth factors added thereafter.

[0032] In the present process according to the invention, 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.

[0033] BRIEF DESCRIPTION OF THE INVENTION

[0034] 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.

[0035] In the present invention 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.

[0036] The invention relates to the following embodiments.

[0037] 1. A method for the preparation of a crosslinked gelatin hydrogel, wherein

[0038] - a cross-linkable gelatin matrix is provided, said matrix comprising primary amine-groups, wherein preferably cross-linkable gelatin matrix is provided in a form (shape or mould),

[0039] - said lyophilized gelatin matrix is permeated by an aqueous crosslinker solution comprising

[0040] - 0.05M to 5M, 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,

[0041] (alternatively at pH 11 to 14 or above, or pH 12 to 14, pH 13 to 14, highly preferably about at pH 12) and

[0042] - 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),

[0043] - allowing the solid lyophilized gelatine to cross-link at a temperature of not more than 15 °C preferably not more than 10°C, in liquid phase, or preferably 0 to 30°C, preferably 0-15°C, for 8-72 hours, to form a crosslinked gelatin hydrogel,

[0044] - washing the crosslinked gelatin hydrogel, preferably with water, until the unreacted crosslinkers are removed from the solution.

[0045] Preferably the lyophilized gelatin matrix comprises at least 70% gelatine.

[0046] Preferably the lyophilized gelatin matrix is prepared from gelatine only.

[0047] Preferably the lyophilized gelatin matrix comprises at most 30% non-gelatine gel-forming agent. Preferably the non-gelatine gel-forming agent is hyaluronic acid.

[0048] In a preferred embodiment 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.

[0049] In a preferred embodiment the crosslinker is a crosslinker capable of acting on amine groups (amine- crosslinker).

[0050] In a preferred embodiment the crosslinker is an epoxy (polyepoxy) crosslinker.

[0051] In a preferred embodiment the epoxy crosslinker is a diglycidyl crosslinker e.g. as defined below e.g. in paragraph 5 or 6.

[0052] In a further preferred embodiment, the epoxy crosslinking agent is a divinyl crosslinker as defined below e.g. in paragraph 7.

[0053] In a preferred embodiment the crosslinker comprises

[0054] - 0.2 M to 1 M alkali hydroxide, and

[0055] - water soluble crosslinker agent in at most 11%, preferably said water soluble crosslinker agent provided in 1% to 10% (V / V) (3-30 pil per 300 pil volume), wherein the crosslinking reaction is carried out at not more than 15°C for 8-72, preferably 30 to 60 hours, to form a crosslinked gelatin hydrogel.

[0056] In a preferred embodiment the gelatine matrix is prepared from a gelatin solution containing 1 to 10% gelatine.

[0057] Preferably 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.

[0058] Preferably 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.

[0059] Preferably the animal is poultry.

[0060] More preferably the animal is a mammal. Preferably the animal a companion animal. Preferably the animal is livestock.

[0061] Highly preferably the animal is human or the patient is human. 2. The invention also relates to a method for the preparation of a crosslinked gelatin hydrogel preferably according to paragraph 1 , wherein

[0062] - a gelatin solution containing 1 to 10% gelatine is prepared, until a homogenous solution is formed,

[0063] - optionally the gelatine solution is provided in a mould (or to have a shape),

[0064] - 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%,

[0065] - 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

[0066] - the alkali hydroxide in 0.2 M to 1 M concentration, and

[0067] - water soluble crosslinker of 0.5 to 20 % (V / V), preferably 5-15% (V / V) cross-linker agent,

[0068] - allowing the solid lyophilized gelatine to cross-link at a temperature of not more than 15°C, preferably 0 to 15°C, for 8-72 hours, preferably 30 to 60 hours to form a crosslinked gelatin hydrogel scaffold,

[0069] - washing the crosslinked gelatin hydrogel, preferably with water, until the unreacted crosslinkers are removed from the solution.

[0070] Preferably the lyophilized gelatin matrix comprises at least 70%, preferably at least 80%, in particular at least 90% gelatine. In a preferred embodiment the lyophilized gelatin matrix essentially consists of gelatine as a gel-forming agent.

[0071] Preferably the lyophilized gelatin matrix comprises at most 30% non-gelatine gel-forming agent. Preferably the non-gelatine gel-forming agent is hyaluronic acid or nothing.

[0072] Preferably 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.

[0073] Preferably the cross-linking temperature is preferably 0 to 10°C or 4 to 10°C.

[0074] In a preferred embodiment 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.

[0075] In a particular embodiment the invention relates to a method for the preparation of a crosslinked gelatin hydrogel according to the invention, wherein

[0076] - a 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,

[0077] - 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%

[0078] - 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,

[0079] - allowing the solid lyophilized gelatine to cross-link, preferably at a temperature of 0 to 15°C, more preferably at 0 to 10°C, or 4 to 15°C, for 30-60 hours to form a crosslinked gelatin hydrogel,

[0080] - washing the 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.

[0081] In a preferred embodiment the epoxy crosslinker is a water soluble epoxy, preferably a diglycidyl crosslinker.

[0082] 3. The method for the preparation of a crosslinked gelatin hydrogel according to invention, in particular according to any of the previous paragraphs, wherein

[0083] - the gelatin solution is lyophilized to prepare a solid, lyophilized gelatin matrix, wherein the gelatin solution is frozen and freeze dried afterwards,

[0084] - the gelatin solution contains 1 to 10% gelatine or 2 to 8% gelatine,

[0085] - the gelatin solution is lyophilized to prepare a solid, lyophilized gelatin scaffold the water content of the which is below 5 w / w%,

[0086] - 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,

[0087] - 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,

[0088] - washing the crosslinked gelatin hydrogel with water until the unreacted crosslinkers are removed from the solution.

[0089] In an alternative embodiment in the preparation of a crosslinked gelatin hydrogel

[0090] - 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

[0091] 5 w / w%

[0092] - 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).

[0093] 4. The method according to paragraph any of the method defined in paragraphs 1 to 3 wherein the 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.

[0094] 5. The method according to paragraph any of the method defined in paragraphs 1 to 4 wherein 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, ethyl, or preferably R1 is a C4-C20 polyether moiety having the general formula (3) wherein n is 1 to 12, preferably 1 to 4, preferably 1 to 2,

[0095] R3 is selected from ethyl, methyl and H, preferably methyl and H.

[0096] 6. The method according to paragraph 5, wherein the crosslinker is a compound having formula (IV), wherein n is 1 to 12, preferably 1 to 4, more preferably 1 to 2.

[0097] Preferably the gelatin solution contains 2 to 8% gelatin,

[0098] - preferably the gelatin solution is lyophilized to reach a water content lower than 5 w / w% of the matrix,

[0099] - 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,

[0100] - the crosslinked gelatin hydrogel is washed, preferably with water.

[0101] Preferably, the method according to paragraph 5, wherein during the crosslinking reaction the crosslinker is conjugated to the gelatin chain in the form of formula (Iva)

[0102] 7. The method according to any of paragraphs 1 to 6 wherein the crosslinked gelatin hydrogel is a crosslinked gelatin hydrogel scaffold preferably for implantation.

[0103] The method according to any of paragraphs 1 to 6 wherein the swelling ratio of the gelatin scaffold is between 5 and 50, preferably between 10 and 30.

[0104] Preferably during crosslinking reaction 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.

[0105] 8. The method according to any of paragraphs 1 to 7 wherein the wherein the temperature is 0 to 10°C (which is preferred) or 4 to 25°C and the reaction time is 24 to 72 hours, preferably 30 to 60 hours, particularly preferably 48 hours and the crosslinker is as defined in any of claims 5 to 6, preferably as defined in paragraph 5 or 6, more preferably paragraph 6.

[0106] In an alternative embodiment the crosslinker is as defined in any of claims 5 to 6. wherein n is 1 to 4, more preferably 1 to 2.

[0107] 9. The method according to any of paragraphs 1 to 8 wherein the lyophilized gelatin scaffold is provided in a form (e.g. mould) and is permeated by an aqueous crosslinker solution in said form.

[0108] Preferably, the gelatin solution is provided by 3D printing.

[0109] The method according to any of paragraphs 1 to 8 wherein the lyophilized gelatin scaffold is provided in a form by 3D printing of the gelatin.

[0110] 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.

[0111] Products

[0112] 10. A crosslinked gelatin hydrogel product,

[0113] (or 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. Preferably said gelatin hydrogel product maintains the porosity of the freeze-dried gelatine matrix but cross-linked. Preferably said crosslinked gelatin hydrogel product has a solid porous material.

[0114] Preferably the crosslinked freeze-dried gelatine matrix is obtained by a method according to any of paragraphs 1 to 9.

[0115] Preferably the crosslinked gelatin hydrogel product is capable of withstanding heat sterilization.

[0116] Preferably the crosslinked gelatin hydrogel product is a solid porous material, preferably forming a solid porous body.

[0117] 11. The crosslinked gelatin hydrogel product according to paragraph 10,

[0118] (or 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.

[0119] Preferably in the crosslinked gelatin hydrogel product of the invention 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.

[0120] 12. The crosslinked gelatin hydrogel product according to any of paragraphs 10 to 11,

[0121] (or a soft tissue implant comprising the crosslinked gelatin hydrogel product), wherein the swelling ratio is 5 to 50, preferably 10 to 30, said swelling ratio being calculated as follows: Swelling ratio= Wswollen gel / Wfreeze-dried gel

[0122] 13. The crosslinked gelatin hydrogel product according to any of paragraphs 10 to 12, wherein the product is in the form of a piece of product having a volume of at most 100 ml.

[0123] In a preferred embodiment 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).

[0124] In a preferred embodiment the piece of crosslinked gelatin hydrogel product is block shaped.

[0125] In a preferred embodiment the piece of crosslinked gelatin hydrogel product has a shape provided by a mould or form as defined in paragraph 11.

[0126] In a preferred embodiment the piece of crosslinked gelatin hydrogel product can be cut into an appropriate size.

[0127] 14. The crosslinked gelatin hydrogel product according to any of paragraphs 10 to 13, wherein the gelatine amino acid with amine side chains are crosslinked by a crosslinker as defined in any of paragraphs 5 to 6.

[0128] Preferably said crosslinked gelatin hydrogel product is obtained by a method of any of paragraphs 5 to 6. 15. The crosslinked gelatin hydrogel product according to claim 14 wherein the crosslinked gelatine has a formula wherein Q and X are, independently, amine nitrogens of the same or different gelatin chain(s), wherein R1 is C4-C50, preferably C4-C20, polyether moiety (having at least two ether oxygens); preferably R1 is a C4-C20 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, more preferably a C2-C4 alkyl), C2-C18 alkylether, a C2-C18 alkeneether, a C2-C18 alkyne (preferably a C2-C8, more preferably a C2-C4 alkyl), preferably R2 is selected from the group consisting of butyl, isopropyl, ethyl, or preferably R1 is a C4-C20 polyether moiety having the general formula (3) wherein n is 1 to 12, preferably 1 to 4, preferably 1 to 2,

[0129] R3 is selected from ethyl, methyl and H, preferably methyl and H, and

[0130] X is a gelatine chain linked via an amine bond, and

[0131] Q is selected from the group of a gelatine chain and a hyaluronic acid chain.

[0132] 16. The crosslinked gelatin hydrogel product according to any of paragraphs 9 or 10 to 15, wherein the lyophilized gelatin scaffold is provided in a form (e.g. mould) and is permeated by an aqueous crosslinker solution in said form.

[0133] Preferably, the crosslinked gelatin hydrogel product is a soft tissue implant.

[0134] Preferably, the gelatin solution is provided by 3D printing. Preferably, the lyophilized gelatin scaffold is provided in a form by 3D printing of the gelatin. Preferably the crosslinked gelatin hydrogel product is supplied with cells, preferably serves as a scaffold for cells.

[0135] 17. A piece (or portion) of a crosslinked gelatin hydrogel product according to any of paragraphs 11 to

[0136] 18, said piece having at most 100 ml. Preferably said piece (or portion) of crosslinked gelatin hydrogel product has a shape. Preferably said piece (or portion) of crosslinked gelatin hydrogel product is a solid porous body.

[0137] 18. 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.

[0138] Preferably, the soft tissue implant comprises a piece of a crosslinked gelatin hydrogel product according to the invention.

[0139] Preferably, 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.

[0140] 19. The soft tissue implant according to paragraph 18, said implant comprising a plurality of scaffolds arranged in a multi-layer stacked configuration.

[0141] 20. The crosslinked gelatin hydrogel as defined in any of claims 10 to 15, for use in the treatment of a patient in need of a soft tissue implant.

[0142] Preferably, said patient is treated by surgery and said crosslinked gelatin hydrogel is used as a filler. Preferably, said patient is treated by a correction surgery after an injury.

[0143] Preferably, said patient is injured in a soft tissue and having a malformation or a soft tissue damage. Preferably, said patient has a developmental disorder wherein a tissue is damaged or a tissue part is missing.

[0144] Preferably, said crosslinked gelatin hydrogel is implanted into or adjacent to an extracellular matrix.

[0145] 21. The use of 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. Preferably, said patient is treated by surgery and said crosslinked gelatin hydrogel is used as a filler.

[0146] 22. Use of a soft tissue implant according to paragraph as defined above, or in any of paragraphs 9 to 15, for promoting tissue regeneration in a patient in need thereof.

[0147] Preferably the patient is a vertebrate patient. Preferably the vertebrate patient is an amphibian, a reptile, a bird, a fish or a mammal. Preferably the patient is a mammalian, highly preferably a human patient.

[0148] 23. A method for use of the soft tissue implant for implantation for treatment of a tissue defect and injury, said method comprising steps: administering said implant into the site of injury in the patient.

[0149] Preferably the patient is a vertebrate patient. Preferably the vertebrate patient is an amphibian, a reptile, a bird, a fish or a mammal. Preferably the patient is a mammalian, highly preferably a human patient. Preferably, 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

[0150] Preferably, said patient is treated by a correction surgery after an injury.

[0151] Preferably, said patient is injured in a soft tissue and having a malformation or a soft tissue damage. Preferably, said patient has a developmental disorder wherein a tissue is damaged or a tissue part is missing.

[0152] Preferably, said crosslinked gelatin hydrogel is implanted into or adjacent to an extracellular matrix. Preferably the soft tissue implant is a medical device.

[0153] 24. Preferably the implant is a face implant, an implant for soft tissue injury, e.g. fat tissue injury of muscle tissue injury. Preferably the implant is used in the field of tissue engineering and regenerative medicine (TERM).

[0154] ABBREVIATIONS

[0155] BDDE: butanediol diglycidyl ether

[0156] GEL: gelatin

[0157] DVS: di vinyl sulfone

[0158] EGDE: ethylene glycol diglycidyl ether

[0159] PEGDE: polyethylene glycol diglycidyl ether

[0160] TERM tissue engineering and regenerative medicine

[0161] Nomenclature of samples: vG-yHA-zN

[0162] - x means the quantity (in mg) of gelatin is used when preparing the scaffold,

[0163] - y means the quantity (in mg) of hyaluronic acid is used when preparing the scaffold,

[0164] - z, if present, means the ratio (in N / N%) of the crosslinker,

[0165] - G means gelatin,

[0166] - HA means hyaluronic acid,

[0167] - N, if present, is the type of the cross-linker, e.g. B means BDDE, P means PEGDE,

[0168] - e.g., scaffold 50G-12.5HA: the scaffold was prepared 50 mg gelatin and 12.5 mg hyaluronic acid.

[0169] DEFINITIONS

[0170] The term “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. In a preferred variant 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.

[0171] 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. Such 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. a sequence 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. Such sequences may be obtained from, for example, suitable altered collagen polynucleotide constructs.

[0172] The term gelatin as used in reference to the present invention encompasses both a gelatin material comprising gelatin polypeptides, as well as an individual gelatin polypeptide.A “crosslinking agent” as described herein refers to a composition comprising a crosslinker.

[0173] “Crosslinker” as used herein 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.

[0174] Preferred crosslinking agents useful in the present invention are diglycidyl or epoxy crosslinkers, which are particularly preferred, as well as divinyl crosslinkers.

[0175] Preferably crosslinkers used herein are homobifunctional crosslinkers or heterobifunctional crosslinkers, preferably homobifunctional crosslinkers.

[0176] The terms “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.

[0177] 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.

[0178] A “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.

[0179] 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. Preferably the polymer is gelatin.

[0180] A matrix is a bulk of homogenous solid material, preferably gel material.

[0181] 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.

[0182] 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.

[0183] An alkylether group is an ether wherein both side of the oxygen is an alkyl or an alkylene group.

[0184] The term "administration" (or application of an implant) as used herein 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. When 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.

[0185] The term "subject" as used herein 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. In a particular embodiment 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.

[0186] The term "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. The term "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.

[0187] The term “comprise(s)” or “comprising” or “including” are to be construed herein as having a non- exhaustive meaning and to allow the addition or involvement of further features or method steps or components to anything which comprises the listed features or method steps or components. Such terms can be limited to “consisting essentially of’ or "comprising substantially" which is to be understood as consisting of mandatory features or method steps or components listed in a list, e.g. in a claim, whereas allowing to contain additionally other features or method steps or components which do not materially affect the essential characteristics of the use, method, composition or other subject matter.

[0188] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural references, and should be construed as including the meaning “one or more”, unless the content clearly dictates otherwise. In general, it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0189] BRIEF DESCRIPTION OF THE FIGURES

[0190] 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

[0191] Figure 2.: Sponge-like structure of the scaffold under microscope (Zeiss AXIO Imager. A 1)(A): lOx and (B): 20x magnification.

[0192] Figure. 3.: Surface of the scaffold using BDDE as crosslinker. (A) 50x, (B) lOOx, (C)250x, (D) 500x, (E) 2000x and (F) 5000x magnification. Figure 4.: Surface of the scaffold using DVS as crosslinker. Small cracks are visible on the surface of the matrix, indicated with white arrows. (A) 50x, (B) lOOx, (C)250x, (D) 500x, (E) 2000x and (F) 5000x magnification.

[0193] Figure 5.: Surface of the scaffold using PEGDE as crosslinker. (A) 50x, (B) lOOx, (C)500x, (D) lOOOx, (E) 2000x and (F) 5000x magnification.

[0194] 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.

[0195] 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).

[0196] Figure 8.: Newly formed blood vessels after 1 month (indicated with red arrows). 2x (A), 4x (B), and lOx (C) magnification.

[0197] Figure 9.: Micrographs of HA-GEL after autoclaving. From left to right, top to bottom: 125GEL, 100GEL25HA, 75GEL50HA, 50GEL5HA, 25GEL100HA, 125HA.

[0198] Figure 10.: The degradation of native freeze-dried gelatin, the significance level was p < 0.05. All data are presented as mean ± standard error of the mean (n=3).

[0199] Figure 11.: 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 significance level was p < 0.05. All data are presented as mean ± standard error of the mean (n=3).

[0200] Figure 12.: 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 Img / ml collagenase (XGC) at 230 nm. The significance level was p < 0.05. All data are presented as mean ± standard error of the mean (n=3).

[0201] Figure 13.: 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. The significance level was p < 0.05. All data are presented as mean ± standard error of the mean (n = 3).

[0202] 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.

[0203] Figure 15.: Relative absorbance of native and crosslinked gelatin matrices at 335 nm (free amine content). The absorbances are expressed as relative absorbances compared to native freeze-dried gelatin (n=3).

[0204] 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.

[0205] 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

[0206] Figure 19A.: Optical microscopic image of a gelatin-based cell scaffold, removed from the black 6 male mouse after 3 months.

[0207] Figure 19B.: Optical microscopic image of a gelatin-hyaluronic acid-based cell scaffold, removed from the black 6 male mouse after 3 months.

[0208] 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).

[0209] 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.

[0210] Figure 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. 5 w / w % GEL, crosslinked in 20 pL BDDE and 300 pL 1 w / w % NaOH for 4 weeks (L), 5 w / w % GEL, crosslinked in 40 pL BDDE and 600 pL 1 w / w% NaOH for 4 weeks (IL), 5 w / w % GEL, crosslinked in 20 pL BDDE and 300 pL 1 w / w % NaOH for 12 weeks (III.), (n=4).

[0211] Figure 24.: Hematoxylin and Eosin (H&E) staining of the explants. 5 w / w % GEL, crosslinked in 20 pL BDDE and 300 pL 1 w / w % NaOH for 4 weeks (L), 5 w / w % GEL, crosslinked in 40 pL BDDE and 600 pL 1 w / w % NaOH for 4 weeks (IL), 5 w / w % GEL, crosslinked in 20 pL BDDE and 300 pL 1 w / w % NaOH for 12 weeks (III.), (n=3).

[0212] Figure 25. : Masson and Goldner (MG) staining of the explants. 5 w / w% GEL, crosslinked in 20 pL BDDE and 300 pL 1 w / w % NaOH for 4 weeks (I.), 5 w / w % GEL, crosslinked in 40 pL BDDE and 600 pL 1 w / w % NaOH for 4 weeks (IL), 5 w / w % GEL, crosslinked in 20 pL BDDE and 300 pL 1 w / w % NaOH for 12 weeks (III.), (n=3).

[0213] DETAILED DESCRIPTION OF THE INVENTION

[0214] 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. Surprisingly, 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. Besides these, DVS was also tested, but in the initial experiment the resulting matrix could not withstand heat sterilization. Therefore, the experiments were continued with the diglycidyl agents. (In later experiments appropriate products were obtained with DVS cross-linker as well, however, lower cross-linking temperatures had to be applied, preferably below 15 °C or more preferably below 10°C.)

[0215] While an alkaline hydroxide solution with high pH, e.g., a 1% NaOH solution would completely degrade a non-crosslinked gelatin gel (GEL), e.g., 5% GEL solution in 4 hours, the same concentration of NaOH with diglycidyl crosslinker, e.g., BDDE, successfully crosslinked the previously freeze-dried 5% GEL after 48 hours, e.g., between 0°C and room temperature to reach a crosslinked material that could withstand heat sterilization.

[0216] Assumably, degradation and crosslinking take place simultaneously, thus, 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.

[0217] It was found that concentrations above 5 V / V% BDDE did not further reduce the swelling ratio, so it was not worth going above that, but at 1 and 3 V / V% BDDE the scaffolds degraded by the collagenase enzyme, so 5% seemed highly preferred.

[0218] To test the crosslinked gel product an enzymatic degradation screening was carried out for gels prepared with all the used BDDE ratios, from 1% to 20% BDDE, to see the degradation effect: 5% BDDE containing one was as stable as the higher BDDE containing scaffolds. Thus, based on the results from the collagenase enzyme related degradation, the inventors chose the 1, 3, and 5% BDDE to investigate the changes in the mechanical properties of native and crosslinked GEL. According to the enzymatic degradation measurements 5 V / V% BDDE matrices were the best to investigate the mechanical tests.

[0219] The chemical modification was assessed with FTIR, as well. The crosslinker concentration difference between the 1, 3 and 5 V / V % BDDE didn’t cause the appearance or disappearance of absorbance peaks, thus it can be concluded that the crosslinking has been effective, using all three concentrations.

[0220] The compression and tensile strength were also tested, the tested mechanical parameters showed similarity to the native gelatin samples. However, the scaffolds were water insoluble, resistant to collagenase enzyme and were able to withstand heat sterilization.

[0221] The matrix of the invention was also tested in vitro to see if hMSCs could adhere to and proliferate on the matrix.

[0222] It was shown that the scaffolds of the invention weren’t toxic for MSCs, cells were able to attach to the surface of the scaffolds after 24 hours and were able to proliferate after 168 hours. Thus, 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).

[0223] In more detail, in the examples of the present invention the use of 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. According to the degradation of native GEL in alkaline solutions, it was found that the presence of TRIS was more profound than the most alkaline pH in this setup, which was 12 in an example. This pH degradation screening was conducted to find the optimal pH that allows the cleavage of the epoxy ring so the crosslinking reaction can start with the use of BDDE and PEGDE. The 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.

[0224] In the present examples 5% gelatin was found to be more stable in a NaOH solution of the pH value of 12 than in buffer of carbonate and / or TRIS, but the crosslinked material at pH=12 further degraded during heat sterilization, thus, although a 1 % NaOH solution would completely degrade a 5% GEL solution in 4 hours, it was found that 1 % NaOH with 1, 3, and 5 V / V % BDDE and PEGDE successfully crosslinked the freeze-dried 5 % GEL after 48 hours to reach a crosslinked material that could withstand heat sterilization.

[0225] As to the 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. Thus, apparently 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. In certain embodiments 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.

[0226] Thus, in the present invention a high pH was applied, and for sake of experiment the GEL content and reaction time were fixed to investigate the preferred amount of the crosslinkers. In order to do that, 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.

[0227] To test another degradation method, 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. et al., 2000] (23, 24), and according to the results obtained, 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. As particular examples characterization with the 1, 3, and 5% BDDE containing ones was carried out.

[0228] The FTIR analysis gave a few answers on the crosslinking reaction. In Fig. 5B it can be observed that instead of the amid II band of native GEL at 1533 cm1a new peak appeared in the crosslinked matrices at 1540 cm1with the use of PEGDE. This can be explained with the partial decomposition of the gelatin chains that can involve the disappearance of amide peaks and the absorbances of the gelatin building blocks can appear. It has long been known that the amino acids that build up gelatin are e.g., valine, leucine, isoleucine and aspartic acid [Eastoe, J.E. et al., 1955]. These amino acids have characteristic absorbance between 1502 and 1514 cm1(25). Generally, 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).

[0229] (Scheme 1)

[0230] (Scheme 2)

[0231] Thus, it is probable that the absorbance of the newly formed bond appears at 1540 cm1and not the amide II band is shifted, however, there was no shift when BDDE was used. The 1447 cm1band did not shift thus probably there were no changes in the secondary structure of the gelatin chain [Deflores, L.P., et al, 2009, Derkach, S.R. et al., 2019] (27, 29). It can also be observed that the intensity of the peaks at 2872 and 2934 cm'1, which is the C-H stretching vibrations of the CH2OH groups, has increased compared to the native gelatin. In the amide III region, native gelatin has a peak at 1233 cm ', but in the crosslinked matrices, the peak has decreased, which is probably due to the changes in the secondary structure of gelatin because of the crosslinking [Stani, C., et al., 2020] (30). The increased peak intensity, which appeared at 1079 cm1can be identified as the C-O-C stretching vibration, which is a typical functional group in both crosslinkers, thus this indicated that the crosslinker became covalently bond to the gelatin chain, and due to the longer chain, it is more intensive in the case of PEGDE. The crosslinker concentration difference between the 1, 3 and 5 V / V % BDDE didn’t cause the appearance or disappearance of absorbance peaks, thus it can be concluded that the crosslinking has been effective, using all three concentrations. And based on the results from the collagenase enzyme related degradation, we chose the 1, 3, and 5 % BDDE to investigate the changes in the mechanical properties of native and crosslinked GEL.

[0232] With the quantification of the free amino groups, we found that 3 and 5 V / V % crosslinker effectively crosslinked the free amino groups in gelatin and the result of the mechanical tests showed that both the tensile strength and the compression was similar to native gelatin.

[0233] 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.

[0234] The freeze-drying method chosen in the present invention results in a particular advantage. Most of the time 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. According to the present method, due to freeze-drying the relative surface of the gelatin matrix increased with the sublimation of water, compared to the starting solid gelatin or a gelatin film. Thus, the alkalic solution of the crosslinker effectively reacted with the functional groups in the gelatin chains and crosslinking was also achieved. Besides this, 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.

[0235] In the present invention, 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.

[0236] It has been found that the product so obtained is compressible and this compressibility is generally better than in case of products made by liquid phase crosslinking and precipitation.

[0237] By using freeze-dried and thus porous starting materials porosity is an inherent feature of the scaffolds of the invention. Thus, lyophilization increases the specific surface of the gel. In general, a lower gelatin concentration also increases the specific surface. However, care should be taken to maintain a sufficient gel strength to avoid damage or collapse of the porous starting material. This is also important in product design, i.e. that after crosslinking the obtained product should have an appropriate mechanical strength to be apt for the purpose to be used. Setting these parameters are possible within the idea and scope of the invention and are at hand of a person skilled in the art.

[0238] Surgical procedures to replace or repair damaged tissue is a very often-used technique the development of which is accelerated by the development of the field of tissue engineering (TE). TE aims to regenerate damaged tissues and uses highly porous scaffold biomaterials, which act as templates cell of the organism. O’Brien et al. describe the functional requirements, and types of materials used in TE applications[O'Brien, F.J., 2011] (7).

[0239] Gelatin

[0240] Traditionally, up to the 20thcentury gelatin, a material processed from collagen, was known as a food additive. However, our age saw a vast variety of its applications from photographic materials to its use in regenerative medicine.

[0241] 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.

[0242] 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).

[0243] 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.

[0244] In our case these functional groups are all present in the monomers of gelatin, thus it would be useful to use a crosslinker that has been used for over a decade and has been generally considered to be safe to use as well as it’s degradation products.

[0245] Thus, in the present invention any type of gelatin can be used.

[0246] Basic methods for treatment and processing of gelatin is well known in the art [Mikhailov, O, V., 2023]

[0247] (34)

[0248] Crosslinkers

[0249] The present inventors have found that the use of butanediol diglycidyl ether (BDDE) is safe, the biocompatibility and cell viability did not change with the use of this material.

[0250] Crosslinking may take place between two functional groups of the gelatin chains, which are usually the - OH or the -NH2 groups. In the present invention, the preferred functional groups are the amino groups, and 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).

[0251] In particular, in the present invention 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.

[0252] Preferably 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.

[0253] In a preferred embodiment 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.

[0254] Preferably 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).

[0255] Preferably R2 is selected from the group consisting of butyl, isopropyl, ethyl,

[0256] Alternatively, preferably 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.

[0257] In a particularly preferred embodiment the crosslinker is a compound having formula (IV), wherein n is a number as defined herein, in particular in said description sites.

[0258] Epoxy crosslinkers, in preferred embodiment diglycidyl crosslinkers, are known in the art.

[0259] A possible mechanism of crosslinking by epoxy crosslinkers under alkalic conditions is also described in Jayachandran, B. et al. [Jayachandran, B., 2022], The authors review that the “crosslinking reaction performed under basic conditions was reported to have proceeded by linking the amine groups of hydroxylysine residues of collagen protein that further resulted in a stiff / rigid material, while under acidic conditions, crosslinking occurred through carboxylic acid (COOH) groups of aspartic or glutamic acid residues of the collagen protein that formed a soft / flexible collagen material.” It appears that under basic conditions by the amine group a nucleophilic attack occurs at the less substituted methylene of epoxide with a collagen amine group, preferably of a lysine residue.

[0260] Various Polyethylene glycol) Diglycidyl Ethers can be obtained from several sources e.g. from Polysciences, Inc. (400 Valley Road, Warrington, PA 18976, USA) or Merck

[0261] A wealth of information is provided on diglycidyl ether and their preparation and uses in Patty’s Toxicology, First published: 2 April 2003; Print ISBN: 9780471319436| Online ISBN: 97804711254711 DOI: 10.1002 / 0471125474. For example, Jean, P. A. et al. review Glycidyl ethers, including aliphatic glycidyl ethers, aromatic monoglycidyl ethers, aromatic diglycidyl ethers, polyglycidyl ethers) and precursor epoxy compounds in Patty’s Toxicology. In a preferred embodiment the scaffold of the invention can be characterized by the following particular features. In a preferred product of the invention 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. In a particularly preferred embodiment, 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.

[0262] 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.

[0263] Also, 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.

[0264] 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.

[0265] 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.

[0266] In the present invention, the most preferred compositions were tested both in vitro and in vivo. The crosslinked scaffolds of the invention are suitable for the attachment and proliferation of cells.

[0267] The number and size of the pores were similar after the crosslinking. The SEM images also revealed that the mechanical disadvantages of the scaffold that were produced with the use of DVS are probably due to the small cracks that are also visible on the 50x magnification image, and are in accordance with earlier observations that the structure was generally more rigid. PEGDE and BDDE proved to be preferred crosslinker, whereas PEGDE resulted in an uneven albeit appropriate surface compared to BDDE. Thus, in a few experiments BDDE was the chosen crosslinker to test the in vivo compatibility of the crosslinked scaffolds.

[0268] No inflammation or total degradation was observed either after one or three months. The explants were observed with the use of optical microscopes, to depict the attachment of tissues and the formation of blood vessels. It was observed that the size of the scaffolds didn’t change after one month, or three months, and the attachment of adipose tissue and blood vessel formation inside the scaffold was visible. It is also visible that the inner side of the implants contained more adipose tissue, which is not surprising, taking into account that the vascularization is more effective on the inner side of the implant, thus that is where the remodeling starts from. The vascularization and the attachment of adipose tissue indicates that the implants are biocompatible, which is advantageous in reconstructive surgery.

[0269] The potential in vivo progress was further analyzed with histological measurements. In the case of EvG staining, there was no significant difference between the relative area of the nuclei, which indicates that the amount of cells that were present are similar in each scaffold. However, between the ECM and cellular components, there was significant difference between Group I. and Group II. and between Group I. and Group III.

[0270] 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. Given that 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.

[0271] 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.

[0272] In the following non-limiting Examples the invention is illustrated in more detail. EXAMPLES

[0273] Source of materials

[0274] 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.

[0275] 1. Preparation of gelatin scaffolds

[0276] 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. 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.

[0277] For scaled up experiments, gelatin scaffolds were also prepared at the concentration of 50 mg / ml. For 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. For the 20 ml samples 400 pl BDDE and 6 ml NaOH were used, for the 30 ml samples 600 pl and 9 ml were used. The crosslinking reaction took place for 48 h at 4 °C. The crosslinked gels were washed with 5 ml of RO water thrice and were freeze-dried again to reach the final form.

[0278] 1.1 Preparation of gelatin scaffolds with DVS

[0279] 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.

[0280] 1.3 Preparation of gelatin scaffolds with PEGDE

[0281] Using PEGDE, 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.

[0282] 1.4 SEM imaging of scaffolds prepared with different crosslinkers

[0283] 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.

[0284] Generally, the surface was similar in all cases, and cells were visible on the surface of the matrices. One major difference can be seen as a difference between the epoxy and vinyl crosslinkers, in the case of DVS, small cracks are visible on the surface of the matrix, indicated with white arrows (figure 4). For a better understanding, the number of pores and pore sizes were compared using ImageJ and the results are shown in figure 6.

[0285] As it is visible in the SEM images and was quantified in figure 6. the number of pores was similar with the use of all three crosslinkers, however, the relative mean area of an average pore was the lowest in the case of BDDE, albeit, the difference was not significant.

[0286] Thus, based on our previous experiments, and the in vitro results, the conclusion was that 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. Among the 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.

[0287] After explantation, 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.

[0288] 2. Preparation of GEL-HA scaffolds

[0289] Six 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). samnlemgelatin mhyaluronic acid (mg) (mg)

[0290] 1 125 0

[0291] 2 100 25

[0292] 3 75 50

[0293] 4 50 75

[0294] 5 25 100

[0295] 6 0 125

[0296] Table 1. Composition of the samples

[0297] 2.2 Optical microscopic measurements of HA-GEL

[0298] The HA-GEL lyophilized mixtures were also examined by light microscopy to determine the differences between the gelatin concentrations.

[0299] 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).

[0300] The results are shown in figure 9. The microscopic images also show that gelatin is present as small particles in the structure of the matrices, the higher the concentration of gelatin, the higher the proportion of these particles. In contrast, HA shows a homogeneous distribution in all cases.

[0301] 3. Degradation of native gelatin in different pH values

[0302] Degradation of native lyophilized * matrices from 2 ml 5 % w / w gelatin were measured at 7 different pH values for 168 hours. The used buffers were the following: H2O (pH=7), PBS (pH=8, 0.01M), TRIS (pH=8, 0.01M andpH=9, 0.01M), Na2CO3and NaHCO3solution (pH=10, 0.01M), NaOH (pH = 11, 0.001 M and pH = 12, 0.01M). 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.

[0303] Two-way ANOVA with Tukey’s post hoc test was performed using the software, Prism 7, and the different groups were compared to each other after 168 hours. The significance level was p < 0.05, where * means that p is between 0.01 and 0.05, ** means that p is between 0.01 and 0.001, and *** means that p is lower than 0.001, and data are presented as mean ± standard error of the mean.

[0304] The degradation of native gelatin was lowest in PBS, which was significantly lower compared to all the other groups. The fastest decomposition was observed in the case of TRIS 8 and TRIS9 solutions, the degradation in these buffers were significantly higher compared to all other groups, however there was no significant difference between the degradation in the two TRIS buffer solutions. Surprisingly, there was no significant difference between the degradation in water compared to NaOH (pH = 12) after 168 hours (Fig. 10).

[0305] The experiment was conducted to find out the stability of GEL and to find the optimal solvent or buffer type for the crosslinking and to compare it to the ones found in the scientific literature. The goal of the optimization process was both to find the optimal pH for the crosslinking, which is in the alkalic interval, and to avoid the further degradation of GEL. Figure 10. shows the degradation of native freeze- dried gelatin. According to Fig. 10, the optimal pH was 12, with the use of NaOH, and the optimal reaction time was 48 hours (data not shown). However, from the viewpoint of scaffold development, sterility is a must have, and we planned to use the most convenient method: heat-sterilization. Unfortunately, the crosslinked product that we produced in the pH=12 NaOH solution further degraded during heat sterilization, thus we moved to 1 % NaOH solution which were found to be optimal during our previous work [Hinsenkamp, A., et al. 2021] (14). The addition of 1 % NaOH solution to the GEL samples under the same circumstances lead to the total degradation of the matrices in 4 hours, surprisingly the simultaneous addition of 1% NaOH and BDDE to GEL resulted in a stable, water insoluble matrix after 48 hours, which was able to withstand heat-sterilization without losing integrity or shape. With the use of DVS, we found that the crosslinked matrices were not able to withstand heat sterilization either, the crosslinked matrix partially decomposed. Thus, we moved on with testing 1 % NaOH solution with BDDE, and PEGDE for the use of the production of crosslinked GEL.

[0306] 4. Weight differences and swelling ratio measurement

[0307] For weight difference measurement, the starting freeze-dried and the crosslinked freeze-dried gelatin samples were compared. Whole lyophilized gelatin matrices were weighed using an analytical balance (Wfreeze dned gei). The gelatin quarters were then allowed to swell for 24 hours and were weighed again (Ws„oiien gei). The swelling ratio was calculated using the following formula.

[0308] Swelling ratio— W swollen gel V freeze-dried gel

[0309] Generally, 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. In a preliminary experiment, we found that the swelling ratio did not further decrease above 5 V / V % crosslinker (data not shown). Thus, we measured the starting, and the crosslinked weights, as well as the swelling ratio to find out the optimal crosslinker reagent amount. 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. According to the results, 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.

[0310] The homogeneity of HA-GEL combinations was 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.

[0311] The experimental results showed that samples containing 25-50-75-75-100 and 125 mg HA could not be controlled well because they were too concentrated. Therefore, the proportions were changed increasing the GEL and decreasing the amount of HA. Samples of 1 ml contain 12.5 mg or 25 mg HA and 50 mg GEL in the present experiment. The crosslinked sample containing the more concentrated 25 mg hyaluronic acid had a higher mass, i.e., was able to absorb more water. This can be explained by the fact that the swelling rate is inversely proportional to the degree of crosslinking. In addition, no significant difference was observed between the swelling rates (mg / mg). It can also be observed that the initial and crosslinked masses are similar for 12.5 mg HA, but for 25 mg HA the crosslinked mass is higher than the initial mass. For this reason, the mixture containing 12.5 mg HA was selected.

[0312] Next, the proportions of the biopolymers were determined and the effect of the ratio of alkali to BDDE required for the crosslinking reaction was studied. In another experiment where 1 ml of lyophilized 50 GEL-12.5 HA was the starting material; this scaffold was placed in a vial with a prepared 300 pl 1% NaOH solution and freshly added 20 pl BDDE was added to the cell scaffold and the reaction was allowed to proceed at room temperature for 24 hours. It was then washed three times and lyophilized. Then, the mass of the crosslinked sample was measured and then it was soaked in 10 ml water overnight to obtain wet mass. The ratio of the wet mass to the crosslinked mass is the swelling ratio. For comparison, the same 1 ml lyophilized 50 GEL-12.5 HA sample was prepared except that the 1% NaOH in the prepared vial was 600 pl and the added BDDE was 40 pl.

[0313] Table 2. Swelling ratio data of the sample containing 50.0 mg gelatin and 12.5 mg hyaluronic acid and various amount of crosslinking agent.

[0314] Mean starting weight Mean crosslinked Mean swelling ratio

[0315] 50G-12.5HA

[0316] (mg) weight (mg) (mg / mg)

[0317] 600 / 40 crosslinker 56.23 50.76 28.67

[0318] 300 / 20 crosslinker 57.03 62.83 12.62

[0319] 5. Enzymatic degradation measurement

[0320] Two different investigations were performed. 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. In the second measurement 5 ml 0.2 mg / ml collagenase was added to native and crosslinked * matrices, which contained 1%, 3%, 5%, 10%, 20% BDDE. In both cases, 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.

[0321] Based on the weight differences, 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.

[0322] 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).

[0323] The aqueous samples (GH) showed relatively little decomposition, with low absorbance even after 48 hours. However, there was significant difference between the native gelatin samples in water and in collagenase (GC) solution after 2 hours. From 4 hours, there was significant difference between the GH and GC group and between the GC and crosslinked GEL in collagenase solution (XGC) group, this difference remained significant even after 48 hours. Thus, with this method we demonstrated that the crosslinking is successful and that BDDE is capable of stabilising gelatin properly. However, the crosslinking took place with the use of 20 V / V % BDDE so planned to further investigate the optimal crosslinking parameters, the enzymatic degradation was tested with the use of reduced BDDE and with 0.2 mg / ml collagenase.

[0324] 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. Thus, as the matrices were found to be stable enough, we continued to work with the 1,3, and 5 V / V % BDDE (50G1B, 50G3B, and 50G5B) containing matrix, accepting that the results with 5 V / V % crosslinking agent are relevant in case of higher concentration crosslinkers as well (Fig. 13).

[0325] The mixtures were allowed to crosslink for 24 hours at room temperature. Prior to using Ehrlich’s reagent, a dilution line for calibration using N-acetylglucosamine (NAG) was prepared. The enzymatic degradation of HA-GEL composites were measured for a week. As a starting point, lyophilized samples were used prepared from HA-GEL mixtures as described above, of which slices indicated in Table 3. were added to the enzyme solutions. Table 3. Masses of slices

[0326] Illgelatin Hlhyaluronic acid Ulslices sample

[0327] (mg) (mg)

[0328] 1 125 0 31.4 mg

[0329] 2 100 25 39.0 mg

[0330] 3 75 50 42.2 mg

[0331] 4 50 75 36.4 mg

[0332] 5 25 100 47.5 mg

[0333] 6 0 125 52.0 mg

[0334] 5 mg of hyaluronidase enzyme and 5 ml of phosphate buffered saline (PBS) was added per sample, then stirred at 37 °C for 168 h. After one day, sample 2 and 3 visibly started to decompose, the other structures remained unchanged. Before starting the first spectrophotometric measurement, 50 pl of borate buffer (pH = 9) was added per sample to the HA-GEL samples to be measured, and stirred in boiling water for 3 minutes. Then 25 pl of 96% acetic acid and 25 pl of Ehrlich’s reagent was added to the samples, the reaction took 12 min to complete. The Ehrlich’s reagent must be added in order to detect the degradation rate of HA. 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.

[0335] Table 4. Results of the spectrophotometric measurements at 585 nm wavelength

[0336] Volume Day Absorbance

[0337] Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6

[0338] 1 0.086 0.144 0.100 0.109 0.112 0.092

[0339] 2 0.088 0.194 0.203 0.196 0.175 0.141

[0340] 3 0.089 0.212 0.224 0.227 0.199 0.168

[0341] 50 pl 4 0.097 0.192 0.215 0.204 0.151 0.139

[0342] 5 0.094 0.227 0.235 0.240 0.205 0.188

[0343] 6 0.094 0.202 0.231 0.215 0.193 0.185

[0344] 7 0.107 0.189 0.212 0.209 0.227 0.189

[0345] 8 0.112 0.231 0.242 0.245 0.233 0.227

[0346] The results of a one-week measurement period nicely show the stability of hyaluronic acid for each hybrid mixture, and the initial and final weights of the samples were measured, which are as described in table 3 below: Table 5. Masses of HA-GEL samples

[0347] Sample Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 29.8 36.5 44.2 40.1 52.4 51.9 28.0 25.6 26.0 25.5 43.9 45.1 g) 1.8 10.9 18.2 14.6 8.5 6.8

[0348] The measurement of the first phase HA-GEL composite gave the expected results, by day 8 the samples 2 and 3 were macroscopically degraded, the sample 4 started to minimally disintegrate, while the others were slower to disintegrate, the experiment was repeated three times.

[0349] 6. Reference example: crosslinking in liquid phase

[0350] As a comparison, 1 ml 5% gelatin solution was used and a crosslinking reaction was carried out by adding 20 pl BDDE as crosslinker in 300 pl 1% NaOH solution (pH = 13) for 48 hours at 37°C. No crosslinking reaction was actually observed an no precipitate obtained, thus the gelatin under these circumstances has probably degraded, rather than crosslinked.

[0351] 7. Structure analysis using FTIR spectroscopy

[0352] 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 cm1in the range of 400-4000 cm1.

[0353] To evaluate the FTIR spectra, we located the main characteristic peaks of gelatin, at 1029 and 1233 cm-1the amide III peaks are visible, these are the C-N stretching vibrations coupled to N-H bending. All these experiments are shown in Figure 14. showing the 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. 14A) and 1, 3, and 5 V / V % PEGDE (Fig. 14B) crosslinker containing matrices are visible.

[0354] The peaks at 1447 cm-1and 1533 cm-1are characteristic for the amide II band, which is caused by the deformation of the N-H bond (19). At 1638 cm-1the amid I region can be observed, which is associated with C=O stretching and bending of N-H bonds with minor C-N stretching. At 3262 cm-1the amide A peaks are visible, these are due to OH stretching and N-H vibration [Hassan N. 2021] (20). 8. Free amino acid content

[0355] 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 reaction with 2,4,6- trinitrobenzenesulfonic acid (TNBS, Sigma-Aldrich) was used based on the description of Grover et al. (18) with further changes developed for our purposes, keeping in mind that we were using water insoluble matrices. 10 mg freeze-dried scaffold was used, 1 ml carbonate puffer (pH=10, 0.1 M) and 500 pl TNBS (10 mM) was added to the matrix. This composition was stored at 30 °C for 30 minutes, then 1 ml SDS (10 m / m%) and 500 pl HC1 (1 M) was added to the mixture. The absorbance was measured at 335 nm with the use of a UV - Vis spectrophotometer (Biotek Powerwave XS, Winooski, VT, USA).

[0356] 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 absorbances are expressed as relative absorbances compared to native freeze-dried gelatin (n=3). The free amino groups are expressed as relative absorbances compared to the absorbance of native freeze-dried gelatin.

[0357] 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.

[0358] 9. Compression test

[0359] To test the compressive properties of the scaffolds, compression test was performed using Instron 5566 universal testing machine (Instron, USA). A capacity of 500 N load cell was used, and the speed of the crosshead was set to 1 mm / min. Native and crosslinked 5% BDDE-containing gelatin scaffolds were used and 3 parallel measurements were taken.

[0360] Based on the FTIR results, swelling and degradation tests, we selected the 5 V / V% BDDE as the optimal amount of crosslinker and the macroscopic properties were further evaluated using a compression test to see the mechanical difference between native gelatin and 50G5B. The results of the load deflection measurement are visible in Fig. 16, wherein the Load - Deflection curves of native GEL scaffolds and 5 V / V % BDDE crosslinked GEL (50G5B) are collected. Both native GEL samples and crosslinked samples were tested in triplicates.

[0361] 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. The results were surprising, the native GEL samples were more rigid and the crosslinked 50G5B material was softer and easier to compress, the load - compression diagram was less steep with the use of crosslinked samples. 10. Tensile strength

[0362] Tensile tests were performed on Instron 5566 universal testing machine (Instron, USA) with a capacity of 500 N load cell. The gauge length was set to 12 mm and a crosshead speed of 2 mm / min was used. Three parallel measurements were carried out on each sample.

[0363] In this experiment the mechanical properties of the scaffolds were evaluated. The results are shown on Figure 17., wherein the average maximum tensile load of native GEL scaffolds and 1, 3 and 5 V / V % BDDE crosslinked GEL are indicated. Both native GEL samples and crosslinked samples were tested in triplicates.

[0364] 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.

[0365] 11. Formulation experiments with GEL-HA samples

[0366] In several parallel experiments, it was found that the most suitable composite variations were obtained by mixing liquid phase materials. In all other cases, the samples were heterogeneous and could not be processed. In one case, the amount of starting gelatin at 50 mg / ml was constant, so we tested mixtures of 5% gelatin and hyaluronic acid in liquid form. First, the gelatin is melted at 50 °C, and then pre-measured samples of hyaluronic acid of 75-100 and 125 mg were added. To make them more homogeneous, the samples were centrifuged at 1000 G centrifugal force for 8 min and allowed to homogenize for another 24 h. After one day, they were centrifuged again at 1000 G for 8 min. The next step was lyophilization, which is performed overnight until the samples are completely dry. The whitish discs were then quartered and weighed individually. Next, 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.

[0367] After optimization, another formulation experiment was performed on the scaffolds. In this case, the concentration of hyaluronic acid was fixed i.e., 1.6% hyaluronic acid solution in liquid form. Again, 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. In one particular case, 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. I then varied the amount of BDDE cross-linker, adding 5, 10, and 20 pl to the remaining scaffold quarters. Each set also contained 1 to 1 control sample to which lacked the crosslinking agent. The setup was the same as in the previous experiment and after 24 h the materials were washed with 5 ml / sample of distilled water. After crosslinking, the samples had to be washed a total of three times with distilled water. After each wash, values were measured by spectrophotometry to determine if any of the crosslinking agent had leached out and, if so, to what extent it had changed. The crosslinked masses were then measured and the results compared with the initial results:

[0368] Table 6. Initial and final masses for the formulation experiments mscaffoid (mg) BDDE (pl) minitiai(mg) mfinai(mg)

[0369] 5 10.8 5.3

[0370] 30 10 10.5 7.4

[0371] 20 11.5 10.6

[0372] 5 17.8 13.5

[0373] 40 10 12.8 8.0

[0374] 20 11.6 8.2

[0375] 5 17.5 13.3

[0376] 50 10 18.7 16.0

[0377] 20 20.0 21.0

[0378] Based on the results, we concluded that 5 N / N% BDDE was sufficient for crosslinking (Hinsenkamp A, 2020).

[0379] 12. Cytotoxicity measurement

[0380] After autoclaving, 5 mm slices were cut from the clean samples of scaffolds prepared with BDDE in a sterile chamber for culturing and growth of mesenchymal stem cells. A stem cell medium was prepared first, to successfully transplant the stem cells onto the scaffolds. The steps of the process must be strictly followed because these are living cells that are very sensitive to small changes. Human bone marrow mesenchymal stem cells (hBM-dMSC, later MSC) were used. The cells were stored in liquid nitrogen at -195.8 °C. A selected number of cells from the freezer had to be melted as quickly as possible in a water bath at 37 °C, because ice crystals can damage the cell membrane if melting is slow. The melted cells were plated in stem cell media.

[0381] Components of 100 ml stem cell medium:

[0382] — 10 ml fetal bovine serum (FBS),

[0383] — 89 ml Dulbecco Modified Eagle Medium (DMEM) containing 4.5 g / L glucose and 1-glutamine,

[0384] — 75 pl bovine fibroblast growth factor (bFGF) — 1 ml penicillin-streptomycin (PEST) antibiotic mixture.

[0385] After mixing the components, 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.

[0386] After 1 week, when the cells had grown to the right level, the stem cell medium was removed and washed the cells with phosphate buffered saline (PBS). 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. At the end of the process, a Biirker chamber cell count method was performed, after which the cells could be used for experiments.

[0387] 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. 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).

[0388] In the experiment, 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.

[0389] According to the results, there were no significant difference between the viability after 24 hours and 168 hours of the two optimal matrices. Thus, reduced BDDE crosslinker concentration was tested to see the potential effect on cell attachment and proliferation, additionally PEGDE was also tested in 5 V / V % concentration. These results are depicted in Fig. 21A and Fig. 21B.

[0390] All scaffolds exhibited relatively similar absorbance values both after 24 hours and 168 hours of culturing with no significant differences observed between them. This indicates that all the scaffolds were suitable to support the viability of the cells. The differences between the surfaces were also compared with the use of SEM. In this case BDDE, DVS, and PEGDE were used as crosslinkers in 5 V / V %, cells were seeded on the matrices and were fixed after one week of culturing.

[0391] 13. In vivo implantation experiment 1 a) Implants using 50G5B4°C cell scaffolds:

[0392] 1 mL of 5% gelatin solution was lyophilized and the lyophilized pad was placed in 300 pl 1% NaOH solution and 20 pl of freshly mixed solution of BDDE and allowed to react for 48 h at 4 °C. After 48 h, it was washed 3 times, autoclaved and sliced into 5 mm diameter scones under sterile conditions.

[0393] For in vivo implantation, black6 male mice, approximately 80-90 days old, were used, and 3 implantations were performed in parallel. Primarily gelatin-based crosslinked cell scaffolds were implanted in the back of mice, under dorsal skin. Mice were then kept under observation for 3 months and terminated by cervical dislocation. The implant was removed and examined under an optical microscope. Vascularisation was clearly visible, as illustrated in Figure 8 and 19 A. The appearance of blood vessels and red blood cells is visible in the picture, the slightly yellow color of gelatin is also visible. The image was captured using a Leica M80 microscope at the magnification rate of 1.25. b) Implants using 50G12.5HA scaffolds:

[0394] 1 ml of a solution containing 5 mg gelatin and 12.5 mg hyaluronic acid was lyophilized and the lyophilized pad was placed in 300 ul of 1% NaOH solution and 20 ul of freshly mixed solution of BDDE and allowed to react for 24 hours at room temperature. After 24 hours, it was washed 3 times, autoclaved and sliced into 5 mm diameter scones under sterile booth.

[0395] 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.).

[0396] The appearance of blood vessels and red blood cells is visible in the picture, however, the surface of the scaffold is more intact probably due to the HA content, and the color is more transparent compared to pure GEL. The image was captured using a Leica M80 microscope at the magnification rate of 1.25. 14. In vivo implantation experiment 2

[0397] In this experiment, 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. At the end of the procedure, the surgical site was treated with Betadine and continued to be treated for an additional 3-4 days to promote healing and maintain cleanliness. The 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.

[0398] After the applied implantation period (either 1 or 3 months), 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.

[0399] 14.1 Histological procedures

[0400] The 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.

[0401] 14.2 Microscopic imaging of the explant

[0402] 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.

[0403] After explantation, and initial imaging the samples were dehydrated and embedded in MMA (methyl methacrylate) and stained for further examination. The main findings were that both cells, connective tissue, and ECM was able to infiltrate the scaffolds, as visible in the images below. The formation of blood vessels was also visible, the vessels were indicated with red arrows in Fig. 22.

[0404] To quantify the contents of the slides after staining, ImageJ was used to characterize the relative areas of the specific constituents. According to the EvG staining, the area occupied by cell nuclei is relatively low across all three samples. There is no significant difference between the 4-week and 12-week samples as presented on Fig. 23. The lowest value is observed in sample “II.”. The ratio of the extracellular matrix (ECM) and cells is the highest in sample “I.”, which is significantly higher compared to sample “II.” and “III.”. H&E staining was also evaluated, with this method, the cells and the scaffold area can be quantified (Fig. 24.).

[0405] There was no significant difference between the area of the nuclei and the scaffold in either of the sample groups. MG staining was also tested to quantify the vessels, the results are shown in Fig. 25.

[0406] The presence of 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.

[0407] INDUSTRIAL APPLICABILITY

[0408] 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. Specifically, 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.

[0409] The 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. With the use of poly(ethylene glycol) diglycydil ether (PEGDE) an appropriate scaffold was received but the surface was found to be uneven. 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. In summary, the implants seem to promote blood vessel formation, support the adherence of adipose tissue as confirmed by optical microscopy and histological evaluations. References

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[0445] (1996)

Claims

CLAIMS1. A method for the preparation of a crosslinked gelatin hydrogel, wherein- a lyophilized cross-linkable gelatin matrix is provided, said matrix comprising primary amine-groups, wherein preferably cross-linkable gelatin matrix is provided in a form,- said lyophilized gelatin matrix is permeated by an aqueous crosslinker solution comprising- 0.2 M to 5 M, preferably 0.2 M to 1 M alkali hydroxide (preferably 0.8% to 4.0 % NaOH; pH above 11), and- water soluble epoxy crosslinker, preferably a diglycidyl ether crosslinker agent of 0.5 to 20 % (V / V), preferably 5-15% (V / V), said crosslinker agent being capable of crosslinking primary amine groups of gelatine, (preferably ethylene glycol diglycidyl ether (EGDE), or polyethylene glycol diglycidyl ether (PEGDE), butane-diol diglycidyl ether (BDDE) preferably said water soluble crosslinker agent,- allowing the solid lyophilized gelatine to cross-link at a temperature of not more 15 °C, for 8-72 hours, to form a crosslinked gelatin hydrogel,- remove unreacted crosslinker agents, wherein the lyophilized gelatin scaffold comprises at least 70% gelatine.

2. The method according to claim 1, wherein- a gelatin solution containing 1 to 10% gelatine is prepared, until a homogenous solution is formed,- the gelatine solution is provided in a mould (or to have a shape),- the gelatin solution, optionally having a shape, is lyophilized to prepare a solid, lyophilized gelatin scaffold, until the water content of the matrix is below 10 w / w%,- the solid lyophilized gelatin scaffold is permeated by the crosslinker solution, preferably without a buffering agent, said solution comprising- the alkali hydroxide in 0.2 M to 1 M concentration, and- water soluble crosslinker of 0.5 to 20 % (V / V), preferably 5-15% (V / V) cross-linker agent,- allowing the solid lyophilized gelatine to cross-link at a temperature of not more than 15°C, preferably 0 to 15°C, for 8-72 hours, preferably 30 to 60 hours to form a crosslinked gelatin hydrogel scaffold,- washing the crosslinked gelatin hydrogel scaffold, preferably with water, until the unreacted crosslinker agents are removed from the solution.

3. The method according to claim 2 wherein- 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 scaffold is permeated by or soaked into a crosslinker solution comprising 0,5 to 10% NaOH, preferably 0,8 to 4 % NaOH, without a buffering agent, and a water-soluble crosslinker of 1 to 10 % (V / V) is applied, wherein preferably the crosslinker is epoxy crosslinker.

4. The method according to paragraph any of the method defined in claims 1 to 3 wherein the 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.

5. The method according to any of the method defined in claims 1 to 4 wherein 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 an ether oxygen, preferably 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, ethyl, wherein * is the linking site of the oxirane (ethylene-oxide) group, or preferably R1 is a C4-C20 polyether moiety having the general formula (3)wherein n is 1 to 12, preferably 1 to 4, preferably 1 to 2,R3 is selected from ethyl, methyl and H, preferably methyl and H.

6. The method according to claim 5, wherein the crosslinker is a compound having formula (IV),wherein n is 1 to 12, preferably 1 to 4, more preferably 1 to 2, wherein preferably- the gelatin solution contains 2 to 8% gelatin,- 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 1 to 15 °C, for 30 - 60 hours to form a crosslinked gelatin hydrogel,- the crosslinked gelatin hydrogel is washed, preferably with water.

7. The method according to any of claims 1 to 6 wherein the swelling ratio of the gelatin scaffold is between 5 and 50, preferably between 10 and 30.

8. The method according to any of claims 1 to 7 wherein the wherein the temperature is 0 to 10°C and the reaction time is 30 to 60 hours, particularly preferably 48 hours, the crosslinker solution comprises 0.8 to 4% NaOH and the crosslinker agent is selected from the group consisting of a water-soluble polyepoxy compounds, more preferably EGDE, PEGDE, BDDE, particularly preferably PEGDE and BDDE.

9. The method according to any of paragraphs 1 to 8 wherein the lyophilized gelatin scaffold is provided in a form (e.g. mould) and is permeated by an aqueous crosslinker solution in said form, or the gelatin solution is provided by 3D printing.

10. A crosslinked gelatin hydrogel product, 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, and wherein said gelatin hydrogel product maintains the porosity of the freeze-dried gelatine matrix, wherein preferably said crosslinked gelatin hydrogel product has a solid porous material.Preferably the crosslinked gelatin hydrogel product and consequently the soft tissue implant has a sponge-like structure. wherein preferably the crosslinked freeze-dried gelatine matrix (scaffold) is obtained by a method according to any of claims 1 to 9.

11. The crosslinked gelatin hydrogel product according to claim 10, 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.

12. The crosslinked gelatin hydrogel product according to any of paragraphs 10 to 11, (or a soft tissue implant comprising the crosslinked gelatin hydrogel product), wherein the swelling ratio is 5 to 50, preferably 10 to 30, said swelling ratio being calculated as follows: Swelling ratio= Wswollen gel / Wfreeze-dried gel.

13. The crosslinked gelatin hydrogel product according to any of paragraphs 10 to 12, (or a soft tissue implant comprising the crosslinked gelatin hydrogel product, wherein the product is in the form of a piece of product having a volume of at most 100 ml.

14. The crosslinked gelatin hydrogel product according to any of paragraphs 10 to 13, (or a soft tissue implant comprising the crosslinked gelatin hydrogel product), wherein the gelatine amino acid with amine side chains are crosslinked by a crosslinker as defined in any of claims (or paragraphs) 5 to 6.

15. The crosslinked gelatin hydrogel product according to claim 14(or a soft tissue implant comprising the crosslinked gelatin hydrogel product), wherein the crosslinked gelatine has a formulawherein Q and X are, independently, amine nitrogens of the same or different gelatin chain(s), wherein R1 is C4-C50, preferably C4-C20, polyether moiety (having at least two ether oxygens);preferably R1 is a C4-C20 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, more preferably a C2-C4 alkyl), C2-C18 alkylether, a C2-C18 alkeneether, a C2-C18 alkyne (preferably a C2-C8, more preferably a C2-C4 alkyl), preferably R2 is selected from the group consisting of butyl, isopropyl, ethyl; or preferably R1 is a C4-C20 polyether moiety having the general formula (3)wherein n is 1 to 12, preferably 1 to 4, preferably 1 to 2,R3 is selected from ethyl, methyl and H, preferably methyl and H, andX is a gelatine chain linked via an amine bond, and Q is selected from the group of a gelatine chain and a hyaluronic acid chain.

16. A soft tissue implant, comprising the crosslinked gelatin hydrogel product according to any of paragraphs 9 to 15, and having a shape adapted to an implantation site of a patient.

17. A soft tissue implant comprising a piece of a crosslinked gelatin hydrogel product,OR comprising multiple pieces of crosslinked gelatin hydrogel product according to paragraph 20 and being adapted to the form of the implantation site.

18. The crosslinked gelatin hydrogel as defined in any of claims 10 to 15, for use in the treatment of a patient in need of a soft tissue implant.Preferably, said patient is treated by surgery and said crosslinked gelatin hydrogel is used as a filler. Preferably, said patient is treated by a correction surgery after an injury.Preferably, said patient is injured in a soft tissue and having a malformation or a soft tissue damage. Preferably, said patient has a developmental disorder wherein a tissue is damaged or a tissue part is missing.Preferably, said crosslinked gelatin hydrogel is implanted into or adjacent to an extracellular matrix.

19. The use of 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. Preferably, said patient is treated by surgery and said crosslinked gelatin hydrogel is used as a filler.

20. 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 10 to 15 is provided, the site of implantation is prepared in said patient by surgical means, wherein aPreferably, said patient is treated by a correction surgery after an injury.Preferably, said patient is injured in a soft tissue and having a malformation or a soft tissue damage. Preferably, said patient has a developmental disorder wherein a tissue is damaged or a tissue part is missing. Preferably, said crosslinked gelatin hydrogel is implanted into or adjacent to an extracellular matrix.

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