Crosslinked polymer material

Abstract

The present invention relates to a crosslinked polymer material detectable by non-invasive imaging, comprising a biocompatible polymeric backbone comprising primary groups, linker groups, and imaging groups; wherein at least part of the primary groups are covalently bonded to the linker groups; and wherein at least part of the imaging groups are covalently bonded to each other and / or to one or more of the linker groups. The present invention further relates to a method to prepare said crosslinked polymer material, and uses of said crosslinked polymer material in biomedical applications such as biofabrication, tissue engineering, and medical devices.

Description

[0001] CROSSLINKED POLYMER MATERIAL

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a crosslinked polymer material detectable by non-invasive imaging, comprising a biocompatible polymeric backbone comprising primary groups, linker groups, and imaging groups; wherein at least part of the primary groups are covalently bonded to the linker groups; and wherein at least part of the imaging groups are covalently bonded to each other and / or to one or more of the linker groups. The present invention further relates to a method to prepare said crosslinked polymer material, and uses of said crosslinked polymer material in biomedical applications such as biofabrication, tissue engineering, and medical devices.

[0004] BACKGROUND TO THE INVENTION

[0005] Gelatin is a nature-derived biopolymer material with excellent cell-interactive properties and the potential to form a hydrogel. As a result, the material has become one of the benchmarks in the field of tissue engineering and biofabrication. However, the stability and mechanical properties under physiological conditions of gelatin have to be improved to make it suitable in biomedical applications. The use of photo-crosslinking strategies is of specific interest as these methods are generally characterized by relatively mild conditions allowing cell encapsulation in the hydrogel. Photo-crosslinking moreover allows for structuring the material, for instance in additive manufacturing techniques like stereolithography, digital light processing and two-photon polymerization. An often-reported gelatin derivative in this respect is gelatin-methacrylamide (Gel-MOD or Gel-MA) in which the amine groups of gelatin have been modified using methacrylic anhydride yielding crosslinkable methacrylamides.

[0006] Dragusin et al. (Soft Matter, 8, 9589-9602, 2012) reported that gelatin-methacrylamide (Gel-MA) can be crosslinked in combination with 2-hydroxyethyl methacrylate (HEMA) to provide a crosslinked hydrogel. They however found that the resulting crosslinked hydrogel was not fully biodegradable.

[0007] The fate of hydrogel scaffolds in vivo is until now, in a large majority, of cases monitored via post-mortem histology. There are however reported cases of diagnostic tools to monitor the in vivo behavior of specific hydrogel scaffolds. For example, Li et al. (Biomater. Sci., 8, 3301 -3309, 2020) reported the use of19F magnetic resonance imaging (19F MRI) to monitor the degradation rate and localization of a fluorine-labeled hydrogel scaffold. For this purpose a polyethylene glycol)-b-poly(L-valine) (PEV) polypeptide hydrogel was grafted with a zwitterionic fluorinated alkynyl molecule via ‘click’ chemistry.

[0008] SUBSTITUTE SHEET (RULE 26) A drawback of reported cases of hydrogels, detectable by non-invasive imaging, is leaching out of particles from the hydrogel, therefore making it difficult to assess whether the signal decrease is due to the biodegradation of the hydrogel or due to the particles being released from the hydrogel. Other disadvantages include the mechanical properties of the hydrogel, its biological compatibility and / or its biodegradability.

[0009] SUMMARY OF THE INVENTION

[0010] It is an object of the present invention to provide a crosslinked polymer material that is detectable by non-invasive imaging, which overcomes or improves one or more of the disadvantages of the prior art, or at least offers one or more useful alternatives. In particular, it is an object of the current invention to provide a polymer material that is detectable by non-invasive imaging, having mechanical and swelling properties that match the properties of the tissue to be regenerated.

[0011] It is another object of the present invention to provide a method for preparing said crosslinked polymer material that is detectable by non-invasive imaging, which overcomes or improves one or more of the disadvantages of the prior art, or at least offers one or more useful alternatives.

[0012] Therefore, in a first aspect, the present invention provides a crosslinked polymer material detectable by non-invasive imaging, comprising: a biocompatible polymeric backbone comprising primary groups; linker groups; imaging groups; and wherein at least part of the primary groups are covalently bonded to the linker groups; and wherein at least part of the imaging groups are covalently bonded to each other and / or to one or more of the linker groups.

[0013] According to a preferred embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the crosslinked polymer material is detectable by magnetic resonance imaging (MRI), in particular detectable by19F MRI; wherein the linker groups provide a linker between the primary groups and the imaging groups; wherein crosslinks connecting two or more of the linker groups comprise one or more imaging groups; wherein the imaging groups are covalently bonded to each other and / or to one or more of the linker groups; and wherein the imaging groups comprise one or more fluorine atoms, and are represented by formula (II)

[0014] SUBSTITUTE SHEET (RULE 26) wherein

[0015] Ri is selected from -H, and -CH3;

[0016] X is selected from -O-, and -NR3-;

[0017] R2 is selected from -Ci ealkyl, and -Cs-ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom; and

[0018] R3 is selected from -H, and -Ci salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; or when X is -NR3-, then R2 and R3 together with the N atom to which they are attached form a 5-6 membered heterocycle; wherein said heterocycle is substituted with at least one -F atom.

[0019] It was found that the crosslinked polymer material as defined herein, comprising covalently bound imaging groups comprising one or more fluorine atoms, is detectable by non-invasive imaging, in particular by magnetic resonance imaging (MRI), more in particular by19F MRI. The linker groups thereby provide a linker between the primary groups of the biocompatible polymeric backbone and the imaging groups.

[0020] It is an advantage of the crosslinked polymer material, as defined herein, that the imaging groups, in essence the groups that enable detectability of the material by nuclear magnetic resonance (MRI), are covalently bonded within the structure of the crosslinked polymer material. Therefore, leaching of the imaging groups does not occur, and moreover ensure that the signal from the nuclear magnetic resonance imaging is proportional to the level of material degrading.

[0021] It is a further advantage of the crosslinked polymer material, as defined herein, that it has highly tunable mechanical properties. The described examples illustrate that the storage modulus (G') of the crosslinked polymer material can be significantly enhanced, compared to comparative materials. This tunability expands the potential applications of the developed hydrogels, for instance enabling a broader range of cell types to be cultivated with the materials in tissue engineering applications.

[0022] SUBSTITUTE SHEET (RULE 26) Specifically, for tissue engineering, the crosslinked polymer materials, as defined herein, provide versatility to support various tissue types. For example, they meet the mechanical requirements for soft tissues such as adipose tissue (G' = 0.3-1 .3 kPa), bone osteoid extracellular matrix (G' = 10-15 kPa), and relatively harder tissues like intervertebral discs (G' = 8-93 kPa), among others. Indeed, the crosslinked polymer materials can be used for different tissue engineering applications, such as adipose tissue replacement and reconstruction, bone engineering, bone replacement and bone reconstruction.

[0023] It is a further advantage of the crosslinked polymer material, as defined herein, that it can be shaped by 3D printing, which enables high-resolution fabrication of detailed structures, such as porous scaffolds, and can match any desired design. It is another advantage of the crosslinked polymer materials, as defined herein, that it can be shaped by electrospinning to provide fibres. It is another advantage of the crosslinked polymer material, as defined, that it can be made injectable, for instance by grinding and reswelling of the ground material.

[0024] According to an embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the biocompatible polymer backbone is a biopolymer selected from polypeptides, proteins, polysaccharides, and polynucleic acids; in particular a biopolymer selected from gelatins, collagens, alginates, dextrans, agarose, glycosaminoglycans, chitosans and carrageenans.

[0025] According to an embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the biocompatible polymer backbone is a polymer selected from a polyester, a polyamide, a polyether, and any combination thereof; in particular a polylactone.

[0026] According to an embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the primary groups comprise amine groups and / or hydroxyl groups and / or carboxylic acid groups.

[0027] According to an embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the linker groups comprise a propanoic or a 2-methylpropanoic moiety.

[0028] According to an embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (Ila)

[0029] SUBSTITUTE SHEET (RULE 26) wherein

[0030] Ri is selected from -H, and -CH3;

[0031] R2 is selected from -Ci ealkyl, and -Cs-ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom.

[0032] According to an embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (lib) wherein

[0033] R1 is selected from -H, and -CH3;

[0034] R2 is selected from -Ci ealkyl, and -Cs ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom; and

[0035] R3 is selected from -H, and -Ci salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; or R2 and R3 together with the N atom to which they are attached form a 5-6 membered heterocycle; wherein said heterocycle is substituted with at least one -F atom.

[0036] According to a particular embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein R2 is selected from the list comprising:

[0037] SUBSTITUTE SHEET (RULE 26)

[0038]

[0039] In a second aspect, the present invention provides a method to prepare the crosslinked polymer material as defined herein, the method comprising the steps of: a) providing a biocompatible polymeric backbone comprising primary groups; b) reacting at least part of the primary groups with a pre-linker group introducing agent to covalently bind pre-linker groups comprising at least one C-C double bond with the primary groups; and c) crosslinking at least part of the pre-linker groups with monomers comprising at least one C-C double bond by free radical chain growth polymerization, wherein at least part of the monomers are imaging monomers.

[0040] According to a preferred embodiment, the present invention provides the method as defined herein, wherein the imaging monomers comprise one or more fluorine atoms and are represented by formula (IV)

[0041] SUBSTITUTE SHEET (RULE 26) wherein

[0042] Ri is selected from -H, and -CH3;

[0043] X is selected from -O-, and -NR3-;

[0044] R2 is selected from -Ci ealkyl, and -Cs-ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom; and

[0045] R3 is selected from -H, and -Ci salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; or when X is -NR3-, then R2 and R3 together with the N atom to which they are attached form a 5-6 membered heterocycle; wherein said heterocycle is substituted with at least one -F atom.

[0046] It was also found that the method according to the invention, comprising covalently bonding at least part of the primary groups of the biocompatible polymeric backbone with pre-linker groups comprising at least one C-C double bond, and crosslinking at least part of the C-C double bond comprising pre-linker groups with monomers comprising at least one C-C double bond, wherein at least part of the monomers are imaging monomers, by free radical chain growth polymerization, provides the crosslinked polymer material as defined herein.

[0047] According to an embodiment, the present invention provides the method as defined herein, wherein the primary groups are amine groups and / or hydroxyl groups and / or carboxylic acid groups.

[0048] According to an embodiment, the present invention provides the method as defined herein, wherein the pre-linker group introducing agent in step b) is an acrylating agent, a methacrylating agent, or any combination thereof, thereby providing pre-linker groups comprising acrylic moieties, methacrylic moieties, or any combination thereof.

[0049] According to an embodiment, the present invention provides the method as defined herein, wherein the imaging monomers are represented by formula (IVa) wherein

[0050] SUBSTITUTE SHEET (RULE 26) R1 is selected from -H, and -CH3;

[0051] R2 is selected from -Ci ealkyl, and -Cs ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom.

[0052] According to an embodiment, the present invention provides the method as defined herein, wherein the imaging monomers are represented by formula (IVb) wherein

[0053] Ri is selected from -H, and -CH3;

[0054] R2 is selected from -Ci ealkyl, and -Cs-ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom; and

[0055] R3 is selected from -H, and -Ci salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; or R2 and R3 together with the N atom to which they are attached form a 5-6 membered heterocycle; wherein said heterocycle is substituted with at least one -F atom.

[0056] In a further aspect, the present invention provides a crosslinked polymer material obtainable by the method as defined herein.

[0057] In a further aspect, the present invention provides the crosslinked polymer material as defined herein, or the crosslinked polymer material prepared according to the method as defined herein, for use in human and / or veterinary medicine.

[0058] It was further found that the crosslinked polymer material as defined herein, or the crosslinked polymer material prepared according to the method as defined herein, is particularly suited for use in tissue engineering, biofabrication, and / or as medical device. Without being limited thereto, the crosslinked polymer material may be used for stereolithography, digital light processing, additive manufacturing, 3D-printing, volumetric additive manufacturing, two-photon polymerization, and the like.

[0059] SUBSTITUTE SHEET (RULE 26) BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 , also abbreviated as Fig. 1 , shows a representation of the results of the in vitro degradation analysis performed on selected polymer materials prepared according to example 5 (G2, G3, G6, and G7) and comparative example A (CA1 , and CA2).

[0061] Figure 2, also abbreviated as Fig. 2, shows a representation of the initial computer aided design (Figure 2a) consisting of 500 x 500 pm struts in 5 mm length and with a total scaffold height of 4 mm to mimic the macromorphology of bone tissue; the optical microscopy images (Figure 2b) of an equilibrium swollen (in PBS) hydrogel scaffold prepared according to example 8; and a SEM micrograph (Figure 2c) of the 3D printed hydrogel scaffold after drying.

[0062] Figure 3a, also abbreviated as Fig. 3a, shows a representation of the results of the in situ rheology analysis of different DFEA monomer molar concentrations in a 10 w / v % solution of gel-MA-100 in DMSO (“Gel-MA-100 with DFEA” (ODFEA / HMA) is 0.25, 10.0, or 20.0); compared to a 10 w / v % solution of gel-MA-100 in DMSO, without addition of DFEA (“Gel-MA-100 without DFEA”).

[0063] Figure 3b, also abbreviated as Fig. 3b, shows a representation of the results of the in situ rheology analysis of different TFEA monomer molar concentrations in a 10 w / v % solution of gel-MA-100 in DMSO (“Gel-MA-100 with TFEA” (HTFEA / HMA) is 20.0), compared to a 10 w / v % solution of gel-MA-100 in DMSO or water, both without addition of TFEA (“Gel-MA-100 without TFEA”).

[0064] Figure 4, also abbreviated as Fig. 4, shows a representation of the results of the in vitro analysis of polymer materials prepared according to example 5 (G1 -12).

[0065] Figures 5a, 5b, and 5c, also abbreviated as Fig. 5a, Fig. 5b, Fig. 5c, show a representation of the signal to noise (S / N) ratio evaluated from the19F MRI measurement of polymer materials prepared according to example 5 (G1 -12).

[0066] Figures 6a, 6b, 6c, also abbreviated as Fig. 6a, Fig. 6b, Fig. 6c, show a representation of the results of the cell seeding performed on selected polymer materials prepared according to example 5 (G2, G6, and G10, respectively).

[0067] Figure 7, also abbreviated as Fig. 7, shows a representation of the results of the cell viability analysis performed on selected polymer materials prepared according to example 5 (G2, G3, G6, G7, G10, G1 1 ).

[0068] SUBSTITUTE SHEET (RULE 26) Figure 8, also abbreviated as Fig. 8, is a graphical representation of a crosslinked polymer material as embodied herein

[0069] Figure 9, also abbreviated as Fig. 9, is a graphical representation of the crosslinking step of the method as embodied herein

[0070] DETAILED DESCRIPTION OF THE INVENTION

[0071] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0072] As mentioned hereinbefore, in a first aspect, the present invention provides a crosslinked polymer material detectable by non-invasive imaging, comprising: a biocompatible polymeric backbone comprising primary groups; linker groups; and imaging groups; wherein at least part of the primary groups are covalently bonded to the linker groups; and wherein at least part of the imaging groups are covalently bonded to each other and / or to one or more of the linker groups.

[0073] It was found that the crosslinked polymer material according to the invention, comprising a biocompatible polymeric backbone comprising primary groups, linker groups, and imaging groups; wherein at least part of the primary groups are covalently bonded to the linker groups; and wherein at least part of the imaging groups are covalently bonded to one or more of the linker groups and / or to each other, thereby providing crosslinking of the biocompatible polymeric backbone, may be detectable by non-invasive imaging.

[0074] The crosslinked polymer material as defined herein may be a hydrogel, i.e. a material capable of binding water in its structure, leading to swelling, but it may also be any other polymeric material without the capability of binding water in its structure. In preferred embodiments of the invention, the crosslinked polymer material is a hydrogel.

[0075] When describing the compounds of the invention, the terms used are to be construed in accordance with the definitions provided hereinafter, unless a context dictates otherwise.

[0076] The term "alkyl" by itself or as part of another substituent refers to a fully saturated hydrocarbon

[0077] SUBSTITUTE SHEET (RULE 26) of Formula CxHzx+i wherein x is a number greater than or equal to 1 . Alkyl groups may be linear or branched and may be substituted as indicated herein. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain. Thus, for example, Ci-4alkyl means an alkyl of one to four carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl, i-propyl, butyl, and its isomers (e.g. n-butyl, i-butyl and t-butyl); pentyl and its isomers, hexyl and its isomers, heptyl and its isomers, octyl and its isomers, nonyl and its isomers; decyl and its isomers. Ci-ealkyl includes all linear or branched groups with between 1 and 6 carbon atoms, and thus includes methyl, ethyl, n-propyl, i-propyl, butyl and its isomers (e.g. n-butyl, i-butyl and t-butyl); pentyl and its isomers, hexyl and its isomers.

[0078] The term "optionally substituted alkyl" refers to an alkyl group optionally substituted with one or more substituents (for example 1 to 6 substituents, for example 1 , 2, 3, 4, 5, or 6 substituents, such as 1 , 2, or 3 substituents) at any available point of attachment. When one or more substituents are present, each substituent replaces a hydrogen atom of the alkyl group.

[0079] The term "cycloalkyl" by itself or as part of another substituent refers to a fully saturated cyclic hydrocarbon of Formula CxHzx wherein x is a number greater than or equal to 3. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain. Thus, for example, C3-4cycloalkyl means a cycloalkyl of three to four carbon atoms. Examples of cycloalkyl groups are cyclopropyl, cyclobutyl, 2- methylcyclopropyl, cyclopentyl, 2-, 3-, or 4-methylcyclopentyl, and cyclohexyl.

[0080] An “optionally substituted cycloalkyl” refers to a cycloalkyl having optionally one or more substituents (for example 1 to 6 substituents, for example 1 , 2, 3, 4, 5, or 6 substituents, such as 1 , 2, or 3 substituents), selected from those defined above for substituted alkyl.

[0081] When the suffix "ene" is used in conjunction with an alkyl or cycloalkyl group, hereinafter also referred to as “alkylene” or “cycloalkylene” respectively, it is intended to mean the alkyl or cycloalkyl group as defined herein having two single bonds as points of attachment to other groups.

[0082] The terms "heterocycle" or "heterocyclyl" as used herein by itself or as part of another group refer to non-aromatic, fully saturated or partially unsaturated cyclic groups (for example, 3 to 13 member monocyclic, 7 to 17 member bicyclic, or 10 to 20 member tricyclic ring systems, or containing a total of 3 to 10 ring atoms) which have at least one heteroatom in at least one carbon atom-containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1 , 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and / or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen

[0083] SUBSTITUTE SHEET (RULE 26) heteroatoms may optionally be quaternized. The heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system, where valence allows. The rings of multiring heterocycles may be fused, bridged and / or joined through one or more spiro atoms.

[0084] Non-invasive imaging is known to the skilled person and includes imaging techniques such as magnetic resonance imaging (MRI), positron emission tomography (PET), ultrasound (US), and radiographical imaging (Rl). Radiographical imaging includes imaging techniques such as X-ray imaging, radiography, computed tomography, sciascopy, and skiagraphy.

[0085] In the context of the present invention, the term ‘primary groups’ refers to chemically defined functional groups present in the biocompatible polymeric backbone that can be further functionalized by a chemical and / or an enzymatic reaction. Examples of such primary groups are amine groups, carboxylic acid groups, and hydroxyl groups. In embodiments of the invention, the primary groups comprised in the biocompatible polymeric backbone comprise amine groups and / or hydroxyl groups and / or carboxylic acid groups. In a particular embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the primary groups comprised in the biocompatible polymeric backbone comprise amine groups and / or hydroxyl groups and / or carboxylic acid groups.

[0086] In the context of the present invention, the term ‘imaging groups’ refers to chemically defined moieties comprising atoms, groups, and / or moieties which are susceptible to non-invasive imaging, in particular to chemically defined moieties comprising one or more fluorine atoms. In the context of the present invention, the terms ‘imaging groups’ are in particular ‘imaging groups comprising one or more fluorine atoms’.

[0087] In the context of the present invention, the term ‘linker groups’ refers to chemically defined moieties providing a linker between a primary group of the biocompatible polymeric backbone and at least part of the imaging groups. Consequently, the imaging groups are covalently bonded in the crosslinked polymer material. The imaging groups can form crosslinks connecting two or more of the linker groups of the crosslinked polymer material or they can form grafts on linker groups. Each imaging group may therefore be connected to only a linker group, to two linker groups, to one linker group and one further imaging group, to two further imaging groups, or to one further imaging group.

[0088] Figure 8 provides a graphical representation of an embodiment of the crosslinked polymer material as defined herein, obtained by radical chain-growth polymerization, comprising a polymeric backbone, linker groups connected to the primary groups of the polymeric backbone, and imaging groups connected to each other and / or to the linker groups.

[0089] SUBSTITUTE SHEET (RULE 26) In embodiments of the invention, the crosslinks connecting two or more of the linker groups may comprise one or more imaging groups and optionally other groups, such as 1 , 2, 3, 4, 5, 10, 15, or more imaging groups and optionally 1 , 2, 3, 4, 5, 10, 15, or more other groups.

[0090] It was found that the crosslinked polymer material as defined herein may be detectable by different non-invasive imaging techniques, such as, but not limited to, magnetic resonance imaging (MRI). In useful embodiments of the invention, the crosslinked polymer material is detectable by magnetic resonance imaging (MRI), in particular detectable by19F MRI.

[0091] It was further found that if at least part of the imaging groups comprised in the crosslinked polymer material as defined herein, comprise one or more fluorine atoms, it may render the crosslinked polymer material detectable by19F MRI. Because fluor is considered a monoisotopic element, consisting of19F as the only stable isotope, when reference is made to ‘a fluorine atom’ or ‘fluorine atoms’, it refers to19F atoms, hence making these atoms susceptible to19F MRI.

[0092] The implementation of hydrophobic low molecular or polymeric19F MRI tracers into applications in physiological environment (aqueous environment) as a part of supra-molecular physically or chemically assembled or crosslinked materials results in a change in the solvation of the rather lipophilic fluorinated tracers as well as the relaxation properties of the tracer. The change in solvation and decrease in solvation of the fluorine atoms typically leads to a lower signal-to- noise ratio during19F MRI imaging. The implementation of F-based compounds into non-soluble crosslinked materials therefore poses a risk of inability to detect such a material in aqueous conditions with19F MRI. It was however surprisingly found that crosslinked polymer materials according to the invention, such as DFEA based crosslinked polymer materials described herein, show similar relaxation properties as the linear poly(DFEA) used as a reference. The exemplified crosslinked polymer materials could be safely visualized in depots below 32 mg of the swollen crosslinked polymer material. Moreover, the detection limit was well below 16 mg for some swollen crosslinked polymer materials where the signal to noise ratio (SNR) is plotted as a function of crosslinked polymer material mass (SNR data). Such detection limits ensure detectability of depots even at later biodegradation phases and may enable us to monitor degradation of crosslinked polymer materials at the site of implantation.

[0093] It appeared that improved mechanical properties of the crosslinked polymer material may be obtained, if the biocompatible polymeric backbone comprises amine groups as primary groups, and at least 20 % of the amine groups, relative to the total amount of amine groups in the biocompatible polymeric backbone, are covalently bonded to the linker groups. It further appeared that a larger relative amount of amine groups covalently bonded to the linker groups, such as 40 %, 50 %, 55 %, 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, or even substantially all amine groups, such as 95 %, 99 %, or even 100 %, may lead to further improved mechanical properties

[0094] SUBSTITUTE SHEET (RULE 26) of the crosslinked polymer material. In useful embodiments of the invention, the biocompatible polymeric backbone comprises amine groups as primary groups, wherein at least 20 % of the amine groups, relative to the total amount of amine groups in the biocompatible polymeric backbone, are covalently bonded to the linker groups. In a particular embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the biocompatible polymeric backbone comprises amine groups as primary groups, and wherein at least 20 % of the amine groups, relative to the total amount of amine groups in the biocompatible polymeric backbone, are covalently bonded to the linker groups, preferably at least 30 %, more preferably from 40 to 100 %.

[0095] It also appeared that improved mechanical properties of the crosslinked polymer material may be obtained, if the biocompatible polymeric backbone comprises hydroxyl groups as primary groups, and at least 20 % of the hydroxyl groups, relative to the total amount of hydroxyl groups in the biocompatible polymeric backbone, are covalently bonded to the linker groups. It further appeared that a larger relative amount of hydroxyl groups covalently bonded to the linker groups, such as 40 %, 50 %, 55 %, 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, or even substantially all hydroxyl groups, such as 95 %, 99 %, or even 100 %, may lead to further improved mechanical properties of the crosslinked polymer material. In useful embodiments of the invention, the biocompatible polymeric backbone comprises hydroxyl groups as primary groups, wherein at least 20 % of the hydroxyl groups, relative to the total amount of hydroxyl groups in the biocompatible polymeric backbone, are covalently bonded to the linker groups. In a particular embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the biocompatible polymeric backbone comprises hydroxyl groups as primary groups, and wherein at least 20 % of the hydroxyl groups, relative to the total amount of hydroxyl groups in the biocompatible polymeric backbone, are covalently bonded to the linker groups, preferably at least 30 %, more preferably from 40 to 100 %.

[0096] It further appeared that further improved mechanical properties of the crosslinked polymer material may be obtained, if the biocompatible polymeric backbone comprises both amine groups and carboxylic acid groups as primary groups, especially when substantially all of the amine groups are covalently bonded to the linker groups, and wherein at least 10 % of the carboxyl acid groups, relative to the total amount of carboxylic acid groups in the biocompatible polymeric backbone, are also covalently linked to the linker groups. It further appeared that a larger relative amount of carboxylic groups covalently bonded to the linker groups, such as 15 %, 20 %, 25 %, 30 %, 40 %, 50 %, 60 %, 70 %, 75 %, or more, may lead to. In useful embodiments of the invention, the biocompatible polymeric backbone comprises both amine and carboxylic acid groups as primary groups, wherein substantially all of the amine groups, and at least 10 % of the carboxylic acid groups, relative to the total amount of carboxylic acid groups in the biocompatible polymeric backbone, are covalently bonded to the linker groups. In a more

[0097] SUBSTITUTE SHEET (RULE 26) particular embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the biocompatible polymeric backbone comprises both amine and carboxylic acid groups as primary groups, wherein substantially all of the amine groups, and at least 10 % of the carboxylic acid groups, relative to the total amount of carboxylic acid groups in the biocompatible polymeric backbone, are covalently bonded to the linker groups, preferably from 15 to 75 % of the carboxylic acid groups, more preferably from 20 to 50 % of the carboxylic acid groups.

[0098] It was further found that when the crosslinks connecting two or more of the linker groups are substantially, such as about 90 %, 95 %, 99 %, or even 100 %, composed of imaging groups comprising one or more fluorine atoms, this may lead to an enhanced signal to noise ratio during19F MRI imaging. In an embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the crosslinks connecting two or more of the linker groups are substantially composed of imaging groups comprising one or more fluorine atoms. In a specific embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the crosslinks connecting two or more of the linker groups are fully composed of imaging groups comprising one or more fluorine atoms.

[0099] It was also found that if more than one fluorine atom is present in the imaging groups, this may lead to an enhanced signal to noise ratio during19F MRI imaging. As stated hereinbefore, the imaging groups may comprise one or more fluorine atoms, such as 1 , 2, 3, 4, 5, or more fluorine atoms. In useful embodiments of the invention, at least part of the imaging groups comprises two or more fluorine atoms, such as 2, 3, 4, 5, or more fluorine atoms. In other useful embodiments of the invention, substantially all, such as about 90 %, 95 %, 99 %, and even 100 %, of the imaging groups comprises two or more fluorine atoms, such as 2, 3, 4, 5, or more fluorine atoms. In a specific embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein substantially all, such as about 90 %, 95 %, 99 %, and even 100 %, of the imaging groups comprise two or more fluorine atoms. In a more specific embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein all the imaging groups comprise two or more fluorine atoms.

[0100] It further appeared that if two or more fluorine atoms are present in the imaging groups, and these two or more fluorine atoms are chemically equivalent, this may lead to an enhanced signal to noise ratio during19F MRI imaging. In an embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein if two or more fluorine atoms are present in at least part of the imaging groups, the two or more fluorine atoms are chemically equivalent. In a specific embodiment, the present invention provides the method as defined herein, wherein if two or more fluorine atoms are present in the imaging groups, the two or more fluorine atoms are chemically equivalent.

[0101] SUBSTITUTE SHEET (RULE 26) It also appeared that when the imaging groups comprising one or more fluorine atoms present in the crosslinked polymer material are the same, this may lead to an enhanced signal to noise ratio during19F MRI imaging. In an embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein substantially all, such as about 90 %, 95 %, 99 %, and even 100 %, of the imaging groups comprising one or more fluorine atoms present in the crosslinked polymer material are the same. In a specific embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups comprising one or more fluorine atoms present in the crosslinked polymer material are the same.

[0102] It was further found that the biocompatible polymeric backbone may be any biopolymer known in the art, such as polypeptides, proteins, polysaccharides, and polynucleic acids. In useful embodiments of the present invention as defined herein, the biocompatible polymer backbone is a biopolymer selected from polypeptides, proteins, polysaccharides, and polynucleic acids. Suitable examples of polypeptides and / or proteins are gelatins, and collagens. Suitable examples of polysaccharides are, alginates, dextrans, agarose, glycosaminoglycans, chitosans and carrageenans. In useful embodiments of the present invention as defined herein, the biocompatible polymer backbone is a biopolymer selected from polypeptides, proteins, polysaccharides, and polynucleic acids. In specific embodiments of the present invention as defined herein, the biocompatible polymer backbone is a biopolymer selected from gelatins, collagens, alginates, dextrans, agarose, glycosaminoglycans, chitosans and carrageenans. In a particular embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the biocompatible polymer backbone is a biopolymer selected from polypeptides, proteins, polysaccharides, and polynucleic acids. In a more particular embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the biocompatible polymer backbone is a biopolymer selected from gelatins, and collagens. In another more particular embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the biocompatible polymer backbone is a biopolymer selected from alginates, dextrans, agarose, glycosaminoglycans, chitosans and carrageenans. In a very specific embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the biocompatible polymer backbone is a gelatin.

[0103] It was also found that the biocompatible polymeric backbone may be other polymers known in the art, such as a polyester, a polyamide, a polyether or any combination thereof. In another embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the biocompatible polymer backbone is a polymer selected from a polyester, a polyamide, a polyether, and any combination thereof. In a particular embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the biocompatible polymer backbone is a polylactone. In a more particular embodiment, the present

[0104] SUBSTITUTE SHEET (RULE 26) invention provides the crosslinked polymer material as defined herein, wherein the biocompatible polymer backbone is a polycaprolactone.

[0105] Depending on the polymerization method used to prepare the polymer, such as condensation polymerization, ring-opening polymerization, the stoichiometry of the monomers, and / or the initiators used, the polymer may comprise amine groups and / or hydroxyl groups and / or carboxylic acid groups as primary groups. Without being limited thereto, the primary groups of the synthetic polymers as defined herein are typically present at one or both ends of the polymer. In the particular case of polycaprolactone, the biocompatible polymeric backbone comprises hydroxyl groups and / or carboxylic acid groups as primary groups. In the more particular case wherein a polyol is used as initiator for the ring-opening polymerization of caprolactone, the resulting polycaprolactone comprises hydroxyl groups as primary groups.

[0106] It appeared that linker groups comprising a propanoic or 2-methylpropanoic moiety may lead to improved properties of the crosslinked polymer material. In an embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the linker groups comprise a propanoic or a 2-methylpropanoic moiety. The propanoic or 2-methylpropanoic moiety may be directly connected to the primary groups of the biocompatible polymeric backbone. For example, when the linker groups comprising a propanoic or a 2-methylpropanoic moiety are covalently connected to an amine primary group, they may form a propanamide or 2-methylpropanamide moiety. When the linker groups comprising a propanoic or a 2- methylpropanoic moiety are covalently connected to a hydroxyl primary group, they may form a propanoic ester or 2-methylpropanoic ester moiety. Alternatively, or complementary, the propanoic or 2-methylpropanoic moiety may be indirectly connected to the primary groups of the biocompatible polymeric backbone. For example, when the biocompatible polymeric backbone comprises carboxylic acid groups as primary groups, the propanoic or 2-methylpropanoic moiety comprised in the linker groups may be covalently connected to the carboxylic acid group via an amidoalkyl fragment.

[0107] It further appeared that imaging groups comprising a propanoic or 2-methylpropanoic moiety may lead to improved properties of the crosslinked polymer material. In an embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (I)

[0108] SUBSTITUTE SHEET (RULE 26) wherein

[0109] M represents a chemical moiety comprising one or more atoms, groups, and / or moieties which are susceptible to non-invasive imaging; and

[0110] R1 is selected from -H, and -CH3.

[0111] As mentioned hereinbefore, it was found that if at least part of the imaging groups comprised in the crosslinked polymer material as defined herein, comprise one or more fluorine atoms, the crosslinked polymer material may be detectable by19F MRI. Therefore, in a particular embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (II) wherein

[0112] Ri is selected from -H, and -CH3;

[0113] X is selected from -O-, and -NR3-;

[0114] R2 is selected from -Ci ealkyl, and -Cs-ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom; and

[0115] R3 is selected from -H, and -Ci salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; or when X is -NR3-, then R2 and R3 together with the N atom to which they are attached form a 5-6 membered heterocycle; wherein said heterocycle is substituted with at least one -F atom.

[0116] It was further found that imaging groups comprising a propanoic ester or 2-methylpropanoic ester moiety may particularly lead to improved properties of the crosslinked polymer material. In a more particular embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (Ila)

[0117] SUBSTITUTE SHEET (RULE 26) wherein

[0118] Ri is selected from -H, and -CH3;

[0119] R2 is selected from -Ci ealkyl, and -Cs-ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom.

[0120] In an even more particular embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (Ila) wherein

[0121] R1 is selected from -H, and -CH3;

[0122] R2 is -Ci-ealkyl; wherein said -Ci ealkyl is substituted with one or more substituents independently selected from -F, and -OH; and wherein R2 comprises at least one -F atom.

[0123] It was also found that imaging groups comprising a specific propanoic ester or 2- methylpropanoic ester moiety may particularly lead to improved properties of the crosslinked polymer material. In a specific embodiment the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (Ila’)

[0124] SUBSTITUTE SHEET (RULE 26) wherein

[0125] Ri is selected from -H, and -CH3;

[0126] FU is selected from -H, -CF3, -CH2CHF2, and -CH2CF3; and

[0127] R5 is selected from -CHF2, -CF3, -CH2CHF2, and -CH2CF3.

[0128] It was further found that imaging groups comprising a propanamide or 2-methylpropanamide moiety may particularly lead to improved properties of the crosslinked polymer material. In another more particular embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (lib) wherein

[0129] R1 is selected from -H, and -CH3;

[0130] R2 is selected from -Ci ealkyl, and -Cs ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom; and

[0131] R3 is selected from -H, and -Ci salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; or R2 and R3 together with the N atom to which they are attached form a 5-6 membered heterocycle; wherein said heterocycle is substituted with at least one -F atom.

[0132] It further appeared that specific imaging groups comprising a propanamide or 2- methylpropanamide moiety may particularly lead to improved properties of the crosslinked

[0133] SUBSTITUTE SHEET (RULE 26) polymer material. In a specific embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (lib’) wherein

[0134] Ri is selected from -H, and -CH3;

[0135] R3 is selected from -H, and -Ci-salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; R4 is selected from -H, -CH3, -CH2OH, -CF3, -CH2CHF2, and -CH2CF3; and

[0136] R5 is selected from -CHF2, -CF3, -CH2CHF2, and -CH2CF3.

[0137] In a more specific embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (lib’) wherein

[0138] R1 is selected from -H, and -CH3;

[0139] R3 is selected from -H, and -Ci-salkyl; wherein said -Ci-salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; R4 is -H; and

[0140] R5is -CHF2.

[0141] In an even more specific embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (lib’)

[0142] SUBSTITUTE SHEET (RULE 26) wherein

[0143] R1 is selected from -H, and -CH3;

[0144] R3is selected from -H, -CH3, -CH2CH3, -CH2OH, -CH2CH2OH, -CHF2, -CF3, -CH2CHF2, and -CH2CF3;

[0145] R4 is -H; and

[0146] R5is -CHF2.

[0147] In very specific embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (lib’) wherein

[0148] Ri is selected from -H, and -CH3;

[0149] R3 is -H;

[0150] R4 is -H; and

[0151] R5is -CHF2.

[0152] In another specific embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (lib”)

[0153] SUBSTITUTE SHEET (RULE 26) wherein

[0154] R1 is selected from -H, and -CH3;

[0155] R3 is selected from -H, and -Ci-salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; and

[0156] Re is selected from -CH2-, -CHF-, and -CF2-; and

[0157] Re' is selected from -CHF-, and -CF2-.

[0158] In a more specific embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (lib”) wherein

[0159] Ri is selected from -H, and -CH3;

[0160] R3 is -H; and

[0161] Re and Re' are both -CHF-, or -CF2-.

[0162] In yet another specific embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (lib’”)

[0163] SUBSTITUTE SHEET (RULE 26) wherein

[0164] Ri is selected from -H, and -CH3; and R7 is selected from -CH2-, -CHF-, and -CF2-; and Ry is selected from -CHF-, and -CF2-.

[0165] In a more specific embodiment, the present invention provides the crosslinked polymer material as defined herein, wherein the imaging groups are represented by formula (lib’”) wherein

[0166] R1 is selected from -H, and -CH3; and

[0167] R7 and Ry are both -CHF-, or -CF2-.

[0168] In useful embodiments of the invention, the imaging groups are represented by any one of formula (II), (Ila), (Ila’), (lib), (lib’), (lib”), or (lib’”); wherein R2 is selected from the list comprising:

[0169] SUBSTITUTE SHEET (RULE 26)

[0170] It also appeared that the ratio of imaging groups to linker groups groups may influence the properties of the crosslinked polymer material. In a specific embodiment, the present invention provides the crosslinked polymer materials as defined herein, wherein the molar ratio of imaging groups to linker groups is from 5 to 90, preferably from 5 to 80, more preferably from 10 to 50, even more preferably from 10 to 40, yet even more preferably from 20 to 40.

[0171] As mentioned hereinbefore, in a second aspect, the present invention provides a method to prepare the crosslinked polymer material as defined herein, the method comprising the steps of: a) providing a biocompatible polymeric backbone comprising primary groups; b) reacting at least part of the primary groups with a pre-linker group introducing agent to covalently bind pre-linker groups comprising at least one C-C double bond with the primary groups; and c) crosslinking at least part of the pre-linker groups with monomers comprising at least one C-C double bond by free radical chain growth polymerization, wherein at least part of the monomers are imaging monomers.

[0172] It appeared that at least part of the monomers comprising at least one C-C double bond used for crosslinking at least part of the C-C double bond comprising pre-linker groups in step c) should be imaging monomers comprising atoms, groups, and / or moieties which are susceptible to non-invasive imaging, for the crosslinked polymer material to be detectable by non-invasive imaging.

[0173] SUBSTITUTE SHEET (RULE 26) It further appeared that when substantially all the monomers comprising at least one C-C double bond are imaging monomers comprising atoms, groups, and / or moieties which are susceptible to non-invasive imaging, this may lead to an enhanced signal to noise ratio during non-invasive imaging.

[0174] Figure 9 provides a graphical representation of an embodiment of the method to prepare the crosslinked polymer material as defined herein, more specifically crosslinking step c) of the method. The left part of the figure represents a polymeric backbone, pre-linker groups connected to at least part of the primary groups of the polymeric backbone, and imaging monomers, before the crosslinking reaction; and the right part of the figure represents a crosslinked polymer material, comprising a polymeric backbone, linker groups connected to at least part of the primary groups of the polymeric backbone, and imaging groups connected to the linker groups and / or to each other, after radical chain-growth polymerization, thereby providing crosslinking.

[0175] As a result of the free radical chain-growth polymerization, the pre-linker groups become covalently bonded linker groups, and the imaging monomers become covalently bonded imaging groups. Imaging monomers thereby become covalently bonded to only a linker group, to two linker groups, to one linker group and one further imaging group, to two further imaging groups, or to one further imaging group.

[0176] In a particular embodiment, the present invention provides the method as defined herein, wherein the primary groups comprised in the biocompatible polymeric backbone comprise amine groups and / or hydroxyl groups and / or carboxylic acid groups.

[0177] As mentioned hereinbefore, it was found that at least part of the imaging groups comprised in the crosslinked polymer material as defined herein, should comprise one or more fluorine atoms to render the crosslinked polymer material detectable by19F MRI.

[0178] It was further found that when substantially all the monomers comprising at least one C-C double bond are imaging monomers comprising one or more fluorine atoms, this may lead to an enhanced signal to noise ratio during19F MRI imaging. In an embodiment, the present invention provides the method as defined herein, wherein substantially all, such as about 90 %, 95 %, 99 %, and even 100 %, of the monomers comprising at least one C-C double bond used for crosslinking at least part of the C-C double bond comprising pre-linker groups in step c), are imaging monomers comprising one or more fluorine atoms. In a specific embodiment, the present invention provides the method as defined herein, wherein all the monomers comprising at least one C-C double bond used for crosslinking at least part of the C-C double bond comprising pre-linker groups in step c), are imaging monomers comprising one or more fluorine atoms.

[0179] SUBSTITUTE SHEET (RULE 26) It also appeared that if more than one fluorine atom is present in the imaging monomers, this may lead to an enhanced signal to noise ratio during19F MRI imaging. In an embodiment, the present invention provides the method as defined herein, wherein at least part of the imaging monomers used for crosslinking at least part of the C-C double bond comprising pre-linker groups in step c), comprise two or more fluorine atoms. In a specific embodiment, the present invention provides the method as defined herein, wherein the imaging monomers used for crosslinking at least part of the C-C double bond comprising pre-linker groups in step c), comprise two or more fluorine atoms.

[0180] It further appeared that if two or more fluorine atoms are present in the imaging monomers, and these two or more fluorine atoms are chemically equivalent, this may lead to an enhanced signal to noise ratio during19F MRI imaging. In an embodiment, the present invention provides the method as defined herein, wherein if two or more fluorine atoms are present in at least part of the imaging monomers used for crosslinking at least part of the C-C double bond comprising pre-linker groups in step c), the two or more fluorine atoms are chemically equivalent. In a specific embodiment, the present invention provides the method as defined herein, wherein if two or more fluorine atoms are present in the imaging monomers used for crosslinking at least part of the C-C double bond comprising pre-linker groups in step c), the two or more fluorine atoms are chemically equivalent.

[0181] It also appeared that when substantially all the imaging monomers comprising one or more fluorine atoms used for crosslinking at least part of the C-C double bond comprising pre-linker groups are the same, this may lead to an enhanced signal to noise ratio during19F MRI imaging. In an embodiment, the present invention provides the method as defined herein, wherein substantially all, such as about 90 %, 95 %, 99 %, and even 100 %, of the imaging monomers comprising at least one C-C double bond used for crosslinking at least part of the C-C double bond comprising pre-linker groups in step c), are the same. In a specific embodiment, the present invention provides the method as defined herein, wherein all the imaging monomers comprising at least one C-C double bond used for crosslinking at least part of the C-C double bond comprising pre-linker groups in step c), are the same.

[0182] It was found that imaging monomers comprising an acrylic or a methacrylic moiety may be particularly suited to crosslink at least part of the C-C double bond comprising pre-linker groups by free radical chain growth polymerization. In an embodiment, the present invention provides the method as defined herein, wherein the imaging groups are represented by formula (III)

[0183] SUBSTITUTE SHEET (RULE 26) wherein

[0184] M represents a chemical moiety comprising one or more atoms, groups, and / or moieties which are susceptible to non-invasive imaging; and

[0185] Ri is selected from -H, and -CH3.

[0186] In a particular embodiment, the present invention provides the method as defined herein, wherein the imaging monomers are represented by formula (IV) wherein

[0187] R1 is selected from -H, and -CH3;

[0188] X is selected from -O-, and -NR3-;

[0189] R2 is selected from -Ci ealkyl, and -Cs ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom; and

[0190] R3 is selected from -H, and -Ci salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; or when X is -NR3-, then R2 and R3 together with the N atom to which they are attached form a 5-6 membered heterocycle; wherein said heterocycle is substituted with at least one -F atom.

[0191] It was further found that imaging monomers comprising an acrylate or a methacrylate moiety may be particularly suited to crosslink at least part of the C-C double bond comprising pre-linker groups by free radical chain growth polymerization. In a more particular embodiment, the present invention provides the method as defined herein, wherein the imaging monomers are represented by formula (IVa)

[0192] SUBSTITUTE SHEET (RULE 26) wherein

[0193] Ri is selected from -H, and -CH3;

[0194] R2 is selected from -Ci ealkyl, and -Cs-ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom.

[0195] In an even more particular embodiment, the present invention provides the method as defined herein, wherein the imaging monomers are represented by formula (IVa) wherein

[0196] R1 is selected from -H, and -CH3;

[0197] R2 is -Ci-ealkyl; wherein said -Ci ealkyl is substituted with one or more substituents independently selected from -F, and -OH; and wherein R2 comprises at least one -F atom.

[0198] In a specific embodiment, the present invention provides the method as defined herein, wherein the imaging monomers are represented by formula (IVa’)

[0199] SUBSTITUTE SHEET (RULE 26) wherein

[0200] R1 is selected from -H, and -CH3;

[0201] R4 is selected from -H, -CF3, -CH2CHF2, and -CH2CF3; and

[0202] R5 is selected from -CHF2, -CF3, -CH2CHF2, and -CH2CF3.

[0203] It was further found that imaging monomers comprising an acrylamide or a methacrylamide moiety may be particularly suited to crosslink at least part of the C-C double bond comprising pre-linker groups by free radical chain growth polymerization. In another more particular embodiment, the present invention provides the method as defined herein, wherein the imaging monomers are represented by formula (IVb) wherein

[0204] Ri is selected from -H, and -CH3;

[0205] R2 is selected from -Ci ealkyl, and -Cs ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom; and

[0206] R3 is selected from -H, and -Ci salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; or R2 and R3 together with the N atom to which they are attached form a 5-6 membered heterocycle; wherein said heterocycle is substituted with at least one -F atom.

[0207] In a specific embodiment, the present invention provides the method as defined herein, wherein the imaging monomers are represented by formula (IVb’) wherein

[0208] SUBSTITUTE SHEET (RULE 26) Ri is selected from -H, and -CH3;

[0209] R3 is selected from -H, and -Ci-salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; R4 is selected from -H, -CH3, -CH2OH, -CF3, -CH2CHF2, and -CH2CF3; and

[0210] R5 is selected from -CHF2, -CF3, -CH2CHF2, and -CH2CF3.

[0211] In a more specific embodiment, the present invention provides the method as defined herein, wherein the imaging monomers are represented by formula (I Vb’) wherein

[0212] R1 is selected from -H, and -CH3;

[0213] R3 is selected from -H, and -Ci salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; R4 is -H; and

[0214] R5is -CHF2.

[0215] In an even more specific embodiment, the present invention provides the method as defined herein, wherein the imaging monomers are represented by formula (I Vb’) wherein

[0216] R1 is selected from -H, and -CH3;

[0217] R3is selected from -H, -CH3, -CH2CH3, -CH2OH, -CH2CH2OH, -CHF2, -CF3, -CH2CHF2, and -CH2CF3;

[0218] R4 is -H; and

[0219] R5is -CHF2.

[0220] SUBSTITUTE SHEET (RULE 26) In a very specific embodiment, the present invention provides the method as defined herein, wherein the imaging monomers are represented by formula (IVb’) wherein

[0221] Ri is selected from -H, and -CH3;

[0222] R3 is -H;

[0223] R4 is -H; and

[0224] R5is -CHF2.

[0225] In another specific embodiment, the present invention provides the method as defined herein, wherein the imaging monomers are represented by formula (IVb”) wherein

[0226] R1 is selected from -H, and -CH3;

[0227] R3 is selected from -H, and -Ci salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; and

[0228] Re is selected from -CH2-, -CHF-, and -CF2-; and

[0229] Re' is selected from -CHF-, and -CF2-.

[0230] In a more specific embodiment, the present invention provides the method as defined herein, wherein the imaging monomers are represented by formula (IVb”)

[0231] SUBSTITUTE SHEET (RULE 26) wherein

[0232] R1 is selected from -H, and -CH3;

[0233] R3 is -H; and

[0234] Re and Re’ are both -CHF-, or -CF2-.

[0235] In another specific embodiment, the present invention provides the method as defined herein, wherein the imaging monomers are represented by formula (I Vb’”) wherein

[0236] Ri is selected from -H, and -CH3; and

[0237] R7 is selected from -CH2-, -CHF-, and -CF2-; and Ry is selected from -CHF-, and -CF2-.

[0238] In a more specific embodiment, the present invention provides the method as defined herein, wherein the imaging monomers are represented by formula (I Vb’”) wherein

[0239] R1 is selected from -H, and -CH3; and

[0240] SUBSTITUTE SHEET (RULE 26) R7 and Ry are both -CHF-, or -CF2-.

[0241] In useful embodiments of the invention, the imaging monomers are represented by any one of formula (IV), (IVa), (IVa’), (IVb), (IVb’), (IVb”), or (IVb’”); wherein R2 is selected from the list comprising:

[0242] It was found that any pre-linker group comprising at least one C-C double bond may allow for crosslinking at least part of the pre-linker groups with imaging monomers comprising at least one C-C double bond by free radical chain growth polymerization, wherein at least part of the imaging monomers comprises atoms, groups, and / or moieties which are susceptible to non- invasive imaging. It was however found that acrylic moieties, methacrylic moieties, or any combination thereof, are particularly suited as pre-linker groups comprising at least one C-C double bond and may lead to improved properties of the crosslinked polymer material. In an embodiment, the present invention provides the method as defined herein, wherein the prelinker groups comprising at least one C-C double bond comprise acrylic moieties, methacrylic moieties, or any combination thereof. The acrylic or methacrylic moiety may be directly connected to the primary groups of the biocompatible polymeric backbone. For example, when the pre-linker groups comprising an acrylic or methacrylic moiety are covalently connected to an amine primary group, they may form an acrylamide or methacrylamide moiety. When the prelinker groups comprising an acrylic or methacrylic moiety are covalently connected to a hydroxyl primary group, they may form an acrylate or methacrylate moiety. Alternatively, or complementary, the acrylic or methacrylic moiety may be indirectly connected to the primary groups of the biocompatible polymeric backbone. For example, when the biocompatible

[0243] SUBSTITUTE SHEET (RULE 26) polymeric backbone comprises carboxylic acid groups as primary groups, the acrylic or methacrylic moiety comprised in the pre-linker groups may be covalently connected to the carboxylic acid group via an amidoalkyl fragment.

[0244] It was further found that if the biocompatible polymeric backbone comprises amine groups and / or hydroxyl groups as primary groups, an acrylating agent, a methacrylating agent, or any combination thereof, is particularly suited to introduce the pre-linker groups on the primary groups. In a specific embodiment, the present invention provides the method as defined herein, wherein the biocompatible polymeric backbone comprises amine groups and / or hydroxyl groups as primary groups, and wherein the pre-linker group introducing agent is an acrylating agent, a methacrylating agent, or any combination thereof. In a more specific embodiment, the present invention provides the method as defined herein, wherein the biocompatible polymeric backbone comprises amine groups and / or hydroxyl groups as primary groups, and wherein the pre-linker group introducing agent is acryloyl chloride, methacrylic anhydride, or any combination thereof.

[0245] It was further found that if the biocompatible polymeric backbone comprises carboxylic acid groups as primary groups, the pre-linker groups may be introduced by an aminoalkylacrylate or an aminoalkylmethacrylate via a Fukuyama-Mitsunobu reaction, or a Sheehan-Hess coupling. In a specific example, in the case of a Fukuyama-Mitsunobu reaction, a combination of (2- aminoethyl)methacrylate, triphenylphosphine, triethylamine, and diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD) may be used to introduce the pre-linker groups. In a specific example, in the case of a Sheehan-Hess coupling, a combination of (2- aminoethyl)methacrylate, and 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC) may be used to introduce the pre-linker groups. In a specific embodiment, the present invention provides the method as defined herein, wherein the biocompatible polymeric backbone comprises carboxylic acid groups as primary groups, and wherein the pre-linker group introducing agent is a combination of a carbodiimide and an aminoalkylacrylate or aminoalkylmethacrylate. In a more specific embodiment, the present invention provides the method as defined herein, wherein the biocompatible polymeric backbone comprises carboxylic acid groups as primary groups, and wherein the pre-linker group introducing agent is a combination of (2-aminoethyl)methacrylate, and 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC). In another specific embodiment, the present invention provides the method as defined herein, wherein the biocompatible polymeric backbone comprises carboxylic acid groups as primary groups, and wherein the pre-linker group introducing agent is a combination of an azodicarboxylate, triphenylphosphine, triethylamine, and an aminoalkylacrylate or aminoalkylmethacrylate. In a more specific embodiment, the present invention provides the method as defined herein, wherein the biocompatible polymeric backbone comprises carboxylic acid groups as primary groups, and wherein the pre-linker group

[0246] SUBSTITUTE SHEET (RULE 26) introducing agent is a combination of (2-aminoethyl)methacrylate, triphenylphosphine, triethylamine, and diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD).

[0247] It also appeared that the ratio of imaging monomers to pre-linker groups may influence the properties of the crosslinked polymer material obtainable by the method. In a specific embodiment, the present invention provides the method as defined herein, wherein the molar ratio of imaging monomers to pre-linker groups is from 5 to 90, preferably from 5 to 80, more preferably from 10 to 50, even more preferably from 10 to 40, yet even more preferably from 20 to 40.

[0248] In a further aspect, the present invention provides a crosslinked polymer material obtainable by the method as defined herein.

[0249] In a further aspect, the present invention provides a use of the crosslinked polymer material as defined herein, or the crosslinked polymer material prepared according to the method as defined herein. In an embodiment, the present invention provides the use of the crosslinked polymer material as defined herein, in human and / or veterinary medicine. In a particular embodiment, the present invention provides the use of the crosslinked polymer material as defined herein, for tissue engineering and biofabrication. In another particular embodiment, the present invention provides the use of the crosslinked polymer material as defined herein, as medical device. The present invention further provides a use of the crosslinked polymer material as defined herein, or the crosslinked polymer material prepared according to the method as defined herein, in stereolithography, digital light processing, additive manufacturing, 3D-printing, volumetric additive manufacturing, or two-photon polymerization.

[0250] In yet a further aspect, the present invention provides the crosslinked polymer material as defined herein, for use in human and / or veterinary medicine. In an embodiment, the present invention provides the crosslinked polymer material as defined herein, for use in tissue engineering or biofabrication.

[0251] The crosslinked polymer material as defined herein may for instance be shaped into a porous or non-porous scaffold suitable for implantation. As mentioned hereinbefore, it is an advantage of the crosslinked polymer material, as defined herein, that it can be shaped by different techniques known in the art, such as 3D printing and electrospinning.

[0252] The crosslinked polymer material as defined herein may also be dehydrated, and ground, and reswollen to provide an injectable hydrogel.

[0253] SUBSTITUTE SHEET (RULE 26) The present invention further provides a method for the prophylaxis and treatment of human and veterinary disorders, by administering the crosslinked polymer material according to this invention to a subject in need thereof. Such crosslinked polymer materials are particularly suitable in various fields such as tissue engineering and biofabrication. As such the crosslinked polymer material of the present invention is highly useful in the treatment of several human and veterinary disorders.

[0254] In yet a further aspect, the present invention provides a medical device comprising the crosslinked polymer material as defined herein. Possible examples of medical devices include, but are not limited to, cell carriers, supports and / or scaffolds for tissue engineering purposes; medical implants, such as dental implants, cartilage implants, tendon implants, ligament implants; coatings on existing medical implants; and drug delivery devices.

[0255] The compounds of the present invention can be prepared according to the reaction schemes provided in the examples hereinafter, but those skilled in the art will appreciate that these are only illustrative for the invention and that the compounds of this invention can be prepared by any of several standard synthetic processes commonly used by those skilled in the art of organic chemistry.

[0256] SUBSTITUTE SHEET (RULE 26) EXAMPLES

[0257] Materials

[0258] Gelatin type B, isolated from bovine hides by an alkaline treatment, provided by Rousselot (Ghent, Belgium). Difluoroethylamine (97%) was purchased from Fluorochem (Derbyshire, UK). Methacrylic anhydride, triethylamine (TEA, > 99%), acryloyl chloride (99%, = 400 ppm phenothiazine), 1 -ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC), Dulbecco’s Modified Eagle Medium (DMEM), 10 v / v% fetal bovine serum, 2 v / v% calcein-acetoxymethyl (Ca-AM), propidium iodide (PI), 2,2,2-trifluoroethylacrylate (TFEA), alginic acid sodium salt from brown algae (sodium alginate, medium viscosity) and deuterium oxide were purchased from Sigma- Aldrich (Diegem, Belgium) and used as received. 2-Aminoethyl methacrylate hydrochloride (AEMA'HCI) was obtained from Polysciences (Conches, France). Dimethyl sulfoxide (DMSO) (99.85%) and N-hydroxysuccinimide (98%) (NHS) were purchased from Acros (Geel, Belgium). (2,4,6-trimethylbenzoyl)-phenyl-phosphinic acid ethyl ester was purchased from Speedcure TPO-L (Lambson, UK). Dialysis membranes Spectra / por (MWCO 12 - 14 kDa) were received from polylab (Antwerp, Belgium). Adipose tissue-derived stem cells (ASCs) were purchased from Lonza (Basel, Switzerland)

[0259] Methods

[0260] Rheological monitoring of crosslinking kinetics

[0261] The crosslinking kinetics were measured using a rheometer Physica MCR-301 (Anton Paar, Sint-Martens-Latem, Belgium) with a parallel plate geometry (upper plate diameter of 25 mm). 300 pL of the sample solution was placed between the plates using a gap setting of 0.35 mm. The edges were trimmed and sealed using silicone grease (Bayer, Sigma-Aldrich, Diegem, Belgium) to prevent sample drying. An oscillation frequency of 1 Hz and a strain of 0.1 % were applied. Next, the solutions were monitored during 5 min at 37 °C and afterwards irradiated using UV-A light (15 min, EXFO Novacure 2000 UV light source at 365 nm using an intensity of 100 mW / cm2). Finally, from the data, the gelation time (tgei) was determined as the time at which the storage modulus (G’) exceeded the loss modulus (G”) of the sample.

[0262] Storage (G’) and loss (G”) moduli of polymer material films

[0263] The mechanical properties of all polymer material films (film with 14 mm diameter) were determined using a rheometer Physica MCR-301 (Anton Paar, Sint-Martens-Latem, Belgium) with a parallel plate geometry (diameter of upper plate 15 mm). The storage (G’) and loss (G”) moduli were quantified by means of oscillation rheology (strain 0.1 %, frequency 1 Hz, and normal force 0.6 N). All measurements were performed in triplicate and reported as mean ± SD.

[0264] SUBSTITUTE SHEET (RULE 26) In vitro Magnetic Resonance Imaging (19F MRI) and Spectroscopy (19F MRS)

[0265] To assess the detectability of the polymer materials in19F MRI and19F MRS, a set of phantoms was prepared. Each phantom contained 500, 250, 125, 63, 32, or 16 mg of corresponding polymer material in a plastic Eppendorf tube (1 .5 mL). These polymer materials were then submerged with ultrapure water and sealed. The phantoms were measured at room temperature using an animal 7 Tesla MRI scanner (MR Solutions, Guildford, UK) equipped with a1H / 19F dual whole-body volume coil for rats.19F MR spectra were acquired by a one-pulse FID sequence (bandwidth 200 kHz, number of acquisitions NA = 32). The19F MR images were acquired using a modified gradient echo sequence (TE = 2.5 ms, TR = 200 ms, FA = 50°, NA = 64, matrix 64 x 64, field of view FOV = 36 x 36 mm2, slice thickness 10 mm).

[0266] Cell seeding & cell viability

[0267] The in vitro cell seeding experiments were performed to investigate the biocompatibility of the polymer materials, and whether these materials can support cell growth, according to ISO- 10993. Selected photo-crosslinked and washed polymer materials were sterilized using a 12 h incubation in 70 v / v% ethanol solution followed by 2 h of UV-C irradiation (100 to 280 nm, 15 mW / cm2). Then, these sterilized samples were swollen again at 37°C in sterile phosphate buffered saline (PBS). Subsequently, adipose tissue-derived stem cells (ASCs) were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10 v / v% fetal bovine serum (FBS) and 1 w / v% penicillin / streptomycin under standard incubator conditions (37 °C, 5% CO2) and with a culture medium change occurring twice a week. ASCs at passage 4 were then seeded at a density of 20,000 ASCs / cm2on the sterilized polymer material samples.

[0268] Next, the biocompatibility of these materials was assessed (by observing the cell morphology and through a live / dead staining) at three consecutive time points (days 1 , 3 and 7). After discarding the culture medium from the cell-seeded constructs, 2 w / v% calcein-acetoxymethyl (Ca-AM) I propidium iodide (PI) staining solution in PBS was added. After incubation of the samples in the dark for 10 minutes, the ASCs were visualized through a green fluorescent protein (GFP) and a Texas Red (TxRed) filter of a fluorescence microscope (Olympus 1X81 with Xcellence Pro software). The percentage viability was quantified using FIJI software.

[0269] In both cases, as a control, the bottom of the well-plate is used.

[0270] In vitro degradation

[0271] In vitro degradation analysis was performed to investigate the biodegradability of the polymer materials. As a model enzyme, collagenase IV from Clostridium histolyticum metalloproteinase- 2 (MMP-2; 0.5 to 5.0 FALGPA units / mg solid, >125 CDU / mg solid) was used, which mimics the

[0272] SUBSTITUTE SHEET (RULE 26) in vivo degradation. Briefly, cylindrical samples (0.8 cm radius, 1 .0 cm height) of selected polymer materials were prepared and incubated in Tris-HCI buffer (0.5 mL, 0.1 M, pH 7.4) in the presence of 0.005% (w / v) NaNs and 5 mM CaCIz at 37 °C for 30 min. Afterwards, 0.5 mL of collagenase solution (200 lU / mL) in Tris-HCI buffer was. The degradation at defined time intervals was stopped by cooling the reaction mixtures with ice and adding EDTA solution (0.1 mL, 0.25 M). Next, the crosslinked polymer materials were washed with ultrapure water and freeze-dried. The residual gel fraction (F) was determined according to the following Equation (1 ): where rrid.t is the dry mass of the sample after degradation at time point t and md,o is the initial mass of the dried sample at t = 0. F was determined in 3 independent measurements and reported as mean ± standard deviation (SD).

[0273] Example 1

[0274] Gelatin-methacrylamide (Gel-MA) with a degree of substitution (DS) of 66 % (referred to as gel- MA-70), and gelatin-methacrylamide (Gel-MA) with a degree of substitution (DS) of 96 % (referred to as gel-MA-100) were prepared according to a protocol described by Van Den Bulcke et al. (Biomacromolecules, vol. 1 , no. 1 , pp. 31-38, 2000). The reaction is represented by the following scheme:

[0275] Briefly, 100 g of gelatin type B was dissolved in 1 L phosphate buffer (pH 7.8) at 40 °C. After complete dissolution 1 or 2.5 equivalents of methacrylic anhydride relative to the primary amines present in the (hydroxy-)lysine and ornithine side chains, were added and the mixture was stirred vigorously to obtain a DS of 66, and 96%, respectively. After 1 h, the mixture was diluted using 1 L of double distilled water (DDW) and introduced in dialysis membranes (Spectra / por MWCO 12 - 14 kDa) during 24 h against DDW. After dialysis, the pH of the mixture was adjusted to 7.4 to mimic the natural ECM more closely using NaOH. Gel-MA-70 (DS 66 %) and gel-MA-100 (DS 96 %) were isolated using lyophilization (Christ freeze-dryer Alpha 2-4 LSC).

[0276] SUBSTITUTE SHEET (RULE 26) Gelatin-methacrylamide-aminoethylmethacrylate (Gel-MA-AEMA) with a degree of substitution of 25 % was prepared according to a protocol described by Van Hoorick et al. (Biomacromolecules, vol. 18, no. 10, pp. 3260-3272, 2017). The reaction is represented by the following scheme:

[0277] Briefly, first, the COOH moieties of gel-MA-100, a gel-MA with a DS of 96 %, were converted into NHS esters by reaction with 3-{[(ethylimino)methylidene]amino}-N,N-dimethylpropan-1 - amine (EDC) and 1 -hydroxypyrrolidine-2, 5-dione (NHS) (0.50 and 0.75 equivalents respectively to mol. amount of carboxylic acid moieties, respectively) in dry DMSO (10 w / v % solution of gel- MA-100) under inert atmosphere. Afterwards, the activated NHS esters were mixed with 2- aminoethyl methacrylate hydrochloride (AEMA-HCI, 0.5 equivalents respectively to mol. amount of COOH moieties for obtaining a degree of substitution of 25 %) in the presence of 4-tert- butylbenzene-1 ,2-diol (inhibitor of radicals to prevent polymerization) in dry DMSO. The resulting material was purified by precipitation in acetone, redissolved in DDW and introduced in dialysis membranes (Spectra / por MWCO 12 - 14 kDa) during 24 h against DDW. After dialysis, the pH of the mixture was adjusted to 7.4 to mimic natural ECM more closely using NaOH and gel-MA- AEMA was isolated using lyophilization (Christ freeze-dryer Alpha 2-4 LSC).

[0278] Example 3

[0279] Alginate-methacrylate (Alg-MA) with a degree of substitution (DS) of 60 % (referred to as Alg- MA) was prepared according to a protocol described by Rouillard et al. (Tissue Engineering Part C:Methods, vol. 17, no. 2, pp. 173-179, 2010). The reaction is represented by the following scheme:

[0280] SUBSTITUTE SHEET (RULE 26)

[0281] Sodium alginate was modified as earlier described by addition of three equivalents (with respect to the hydroxyl groups on the alginate backbone) of methacrylic anhydride in order to obtain a high degree of substitution. Briefly, a 2 w / v% sodium alginate solution was prepared by dissolving 20 g of sodium alginate in ultrapure water using a mechanical stirrer. In a next step, methacrylic anhydride was added dropwise to the solution. During the reaction, methacrylic acid was formed as a side product, which lowers the pH of the solution and lowers the efficiency of the reaction. For that reason, the pH was constantly monitored and kept at pH 8 by dropwise addition of a 5 M sodium hydroxide (NaOH). After addition of methacrylic anhydride, the solution was stirred for 24 hours at room temperature. The methacrylated alginate was purified by dialysis (cut-off 12-14 kDa) during 72 hours, changing the dialysis water twice a day to remove unreacted reagents and the formed methacrylic acid. After dialysis, the samples were freeze-dried. The degree of substitution was determined by1H NMR to be 60% of monomeric units modified.

[0282] Example 4

[0283] A / -2,2-difluoroethylacrylamide (DFEA) was prepared via a reaction of 2,2-difluoroethylamine with acryloyl chloride and triethylamine in tetrahydrofuran at room temperature, as described by Kolouchova et al. (Journal of Controlled Release, vol. 327, pp. 50-60, Nov. 2020).

[0284] Briefly, 2,2-difluoroethylamine was mixed with triethylamine (1 .2 eq.), dissolved in dry tetrahydrofuran (THF, 4 wt.% solution of 2,2-difluoroethylamine) and cooled down to 0 °C. Freshly distilled acryloyl chloride (1 .2 eq.) diluted with dry THF (1 :1 ratio) was added dropwise to the solution of amines upon vigorous stirring at 0 °C and then the reaction was stirred at room temperature for 7 h. Afterwards, the THF solution was concentrated using a rotary evaporator, diluted with diethylether and washed using an aqueous NaHCOs solution (1 M) and saline using a separatory funnel. The organic phases were dried with MgSCk, the solvent was removed using a rotary evaporator, and the product was purified by flash chromatography in multiple batches (mobile phase: ethyl acetate / hexane 1 :1 ). The yield was approx. 70 %.

[0285] SUBSTITUTE SHEET (RULE 26) Example 5

[0286] Polymer materials were prepared by a combination of either one of the gelatin-based materials Sprepared in example 1 (gel-MA-70, gel-MA-100), or example 2 (gel-MA-AEMA), with the DFEA monomer prepared in example 4. The reagents were dissolved in various ratios in dry DMSO at 50 °C. Concentration of gelatin-based material + DFEA monomer was around 10 w / v % for all prepared solutions. These solutions were prepared in dark glass vials and protected from light. Subsequently, Li-TPO-L (8 mg / mL solution in ultrapure water; 2 mol.% relative to the number of MA and AEMA moieties in the reaction mixture) was added to the mixture and flushed with argon for 2 min. Afterwards, the solution was injected between two parallel glass plates (molds) separated by a 1 mm thick silicone spacer and covered with a thin poly(tetrafluoroethylene) sheet. Next, these molds were irradiated from both sides with UV-A light (365 nm, 100 mW / cm2) for 1 h.

[0287] After the photopolymerization, the polymer material films were washed (ethanol / water 70:30 (v / v) mixture) for 24 h (solvent was replaced with fresh solvent at least 5 times during 24 h) and subsequently in ultrapure water or PBS (140 mM, pH = 7.4) for another 24 h (solvent was replaced with fresh solvent at least 5 times during 24 h).

[0288] Table 1 provides an overview of polymer materials prepared according to example 5; mgeiis the mass of the corresponding starting material; HMA + AEMA is the molar amount of MA and AEMA moieties in the reaction mixture; ITIDFEA is the mass of DFEA in the reaction mixtures; ODFEA is the molar amount of DFEA in the reaction mixtures; cgeiis the concentration of the corresponding gelatin-based starting materials; and ODFEA / OMA+AEMA is the ratio of molar amounts of DFEA vs MA+AEMA moieties, where applicable, in the reaction mixtures.

[0289] The suitability of the polymer materials to serve tissue engineering applications is determined by their swelling and rheological properties, as well as the storage modulus G’ of the polymer materials, since this parameter determines the polymer materials’ suitability for supporting adhesion and cell proliferation of various cell types.

[0290] Table 1. Specification of reaction mixtures according to Example 5

[0291] Cgel mgeiHJDFEA HOFFA

[0292] Code Starting material (w / v o / (mg) (mg) / HMA+AEMA

[0293] G1 gel-MA-70 8.2 147.1 50.0 10

[0294] G2 gel-MA-70 6.6 147.1 100.0 20

[0295] G3 gel-MA-70 4.7 147.1 200.0 40

[0296] G4 gel-MA-70 3.0 147.1 400.0 80

[0297] SUBSTITUTE SHEET (RULE 26) G5 gel-MA-100 7.4 100.0 50.0 10

[0298] G6 gel-MA-100 5.5 100.0 100.0 20

[0299] G7 gel-MA-100 3.4 100.0 200.0 40

[0300] G8 gel-MA-100 2.0 100.0 400.0 80

[0301] G9 gel-MA-AEMA 5.9 57.6 50.0 10

[0302] G10 gel-MA-AEMA 4.0 57.6 100.0 20

[0303] G11 gel-MA-AEMA 2.4 57.6 200.0 40

[0304] G12 gel-MA-AEMA 1.3 57.6 400.0 80

[0305] Table 2 shows analysis data of the crosslinked gelatin-DFEA polymer materials prepared according to example 5: calculated ratio between the MA, AEMA and DFEA monomers, storage modulus (G’), swelling ratio (SR), and gel fraction (GF).

[0306] It can be seen from Table 2 that the amount of DFEA and MA, in the reaction mixture to prepare the crosslinked polymer materials, can be used to fine-tune the fluorine content and swelling ratios of the crosslinked polymer materials. In gel-MA-70- and gel-MA-100-based polymer materials, when the DFEA content is increased, the swelling ratio (SR) increases as well. In contrast, the SR of the gel-MA-AEMA-based materials is relatively constant regardless of the DFEA content and generally lower than for the gel-MA-70- and gel-MA-100-based materials. Without willing to be bound by any theory, this may be attributed to the high crosslink density of the gel-MA-AEMA-based materials.

[0307] Table 2. Analysis data of polymer materials prepared according to Example 5 rtDFEA / OMA in the G’

[0308] Code SR ± SD GF ± SD (%) polymer (kPa) material

[0309] G1 9.5 ±0.37 4.8 ±1.0 11.9 ± 1.3 74.2 ± 7.5

[0310] G2 12.6 ±0.26 7.8 ±0.8 13.4 ±1.3 71.0 ±6.2

[0311] G3 15.5 ±0.26 5.5 ±0.7 14.9 ±0.2 72.1 ±0.9

[0312] G4 16.6 ±0.17 3.8 ±0.4 16.6 ±0.8 60.3 ± 3.2

[0313] G5 7.4 ±0.23 8.8 ±1.7 8.5 ± 0.9 70.2 ±5.1

[0314] G6 9.8 ±0.18 11.9 ±1.9 12.2 ±0.6 71.3 ±0.1

[0315] G7 11.4±0.14 7.1 ±1.8 18.1 ±2.3 66.5 ± 2.7

[0316] G8 12.0 ±0.16 3.6 ±0.2 14.6 ±0.7 58.5 ± 2.0

[0317] G9 3.5 ±0.14 6.0 ±1.0 9.9 ± 0.7 65.5 ± 0.8

[0318] G10 5.9 ±0.11 3.4 ±0.3 11.8 ± 1.2 65.2 ± 3.2

[0319] G11 7.2 ±0.09 2.4 ±0.4 7.7 ± 1.9 65.0 ±2.1

[0320] SUBSTITUTE SHEET (RULE 26) G12 5.0 ± 0.13 1 .9 ± 0.1 7.2 ± 0.3 59.3 ± 1 .5

[0321] It was further found that the DFEA content in the polymer material can be changed, and hence the amount of fluorine in the polymer material, without significant changes to the storage modulus G’ and the swelling ratio (SR). It was also found that the G' values of the polymer materials prepared in example 5 match the mechanical properties of soft tissues (e.g., adipose tissue, G' = 0.3 to 1 .3 kPa; prostate tissue, G' = 6.6 to 21 .9 kPa; intervertebral discs, G' = 8 to 93 kPa).

[0322] Comparative example A

[0323] Polymer materials without fluorine (CA1 -CA3) were prepared according to example 5 by using either one of the gelatin-based materials prepared in example 1 (gel-MA-70, gel-MA-100), or example 2 (gel-MA-AEMA), respectively, by dissolution in dry DMSO at 50 °C, having a concentration of around 10 w / v % for all three solutions, but without addition of DFEA.

[0324] In vitro degradation analysis was performed to investigate the effect of MA and DFEA content on the biodegradability of selected polymer materials with DFEA (G2, G3, G6, G7) and without DFEA (CA1 , CA2), as described above. Figure 1 shows that crosslinked polymer materials without DFEA (CA1 based on gel-MA-70 and CA2 based on gel-MA-100) and tested gel-MA- 70-based crosslinked polymer materials (G2 and G3) degraded quickly with zero-order kinetics. In contrast, tested gel-MA-100-based crosslinked polymer materials (G6 and G7) exhibited significantly slower, but still zero-order kinetics degradation. In contrast to gel-MA based polymer materials crosslinked with HEMA, as reported by Dragusin et al., it was surprisingly found that gel-MA based polymer materials crosslinked with DFEA may be fully biodegradable. It was moreover found that the DFEA content allows to tune the biodegradability.

[0325] Example 6a

[0326] Polymer materials with fluorine (F1 -F3) were prepared according to example 5 by using either one of the gelatin-based materials prepared in example 1 (gel-MA-70, gel-MA-100), or example 2 (gel-MA-AEMA), respectively, by dissolution in dry DMSO at 50 °C, having a concentration of around 10 w / v % for all three solutions, and subsequently adding DFEA to obtain a molar ratio of DFEA and MA+AEMA moieties in the gelatin-based starting materials in the reaction mixtures of 20. This is in contrast to example 5, wherein all the reaction mixtures solutions have a concentration of gelatin-based material + DFEA monomer of around 10 w / v %.

[0327] Example 6b

[0328] A polymer material with fluorine (TF1 ) was prepared according to example 5 by using the gelatinbased material prepared in example 1 (gel-MA-100) by dissolution in dry DMSO at 50 °C, having

[0329] SUBSTITUTE SHEET (RULE 26) a concentration of around 10 w / v %, and subsequently adding TFEA to obtain a molar ratio of TFEA vs MA moieties in the gelatin-based starting material in the reaction mixture of 20.

[0330] Table 3 provides an overview of polymer materials prepared according to comparative example A and examples 6a and 6b; cgeiis the concentration of the corresponding gelatin-based starting materials; noFEA or TFEA / HMA+AEMA is the ratio of molar amounts of DFEA or TFEA vs MA+AEMA moieties, where applicable, in the gelatin-based starting materials in the reaction mixtures; and G' is the storage modulus of the resulting polymer material.

[0331] Table 3. Specification of reaction mixtures and analysis data of polymer materials prepared according to Comparative example A, Example 6a and Example 6b

[0332] Code Starting material (w / v %) TFEA (kPa)

[0333] / OMA+AEMA

[0334] CA1 Gel-MA-70 10.0 0 4.6 ± 1 .1

[0335] F1 Gel-MA-70 10.0 20 8.2 ± 1 .4

[0336] CA2 Gel-MA-100 10.0 0 7.9 ± 1 .2

[0337] F2 Gel-MA-100 10.0 20 12.2 ± 1 .9

[0338] TF1 Gel-MA-100 10.0 20 13.4 ± 0.8

[0339] CA3 Gel-MA-AEMA 15.0 0 5.1 ± 2.1

[0340] F3 Gel-MA-AEMA 15.0 20 12.3 ± 1 .4

[0341] From Table 3, it can be seen that addition of the DFEA or TFEA monomer to each of the solutions containing a gelatin-based material, results in an increase of the storage modulus G’.

[0342] Example 7

[0343] Polymer materials with fluorine (ATF1 -ATF2) were prepared by using alg-MA prepared according to example 3, by dissolution of alg-MA in ultrapure water, having a concentration of around 5 w / v %, adding TFEA, and adding 2 wt.% of Tween to the mixture to disperse the liquid monomer in the aqueous solution of alg-MA. The materials were crosslinked as described in example 5, and their exact ratios are described in Table 4.

[0344] Comparative example B

[0345] A polymer material without fluorine (CB) was prepared according to example 7 by using alg-MA, but without addition of TFEA, and wherein the concentration of alg-MA in ultrapure water was around 5 w / v %.

[0346] SUBSTITUTE SHEET (RULE 26) Table 4. Specification of reaction mixtures and analysis data of polymer materials prepared according to Example 7 and Comparative example B

[0347] Calg ODFEA or G’

[0348] Code Starting material

[0349] (w / v %) TFEA / rtMA (kPa)

[0350] CB Alg-MA 5.0 0 1 .97 ± 0.5

[0351] ATF1 Alg-MA 5.0 0.1 6.46 ± 1 .9

[0352] ATF2 Alg-MA 5.0 0.2 15.4 ± 2.3

[0353] From Table 4, it can be seen that the mechanical properties of ATF1 and ATF2 show an increase in stiffness compared to the comparative example CB.

[0354] Example 8

[0355] A 3D crosslinked polymer material was prepared by dissolution of 150 mg gel-MA-AEMA, and 260 mg DFEA in the presence of 20 mol% Li-TPO-L and 0.72 mol% tartrazine with regards to the double bonds present in the precursors in 1 mL of dry DMSO. The solution was transferred to the vat of the printer (Cellink LumenX, Gothenburg, Sweden), the printing parameters were set as 19.51 mW / cm2intensity at A = 405 nm, 2.5 s curing time / layer at room temperature. Subsequently, the scaffolds were washed for 24 hours (EtOH:water 70:30) to remove the unreacted moieties and equilibrium swollen in PBS (140 mM pH 7.4) for 24 hours at 37 °C.

[0356] Figure 2 depicts the initial computer aided design (Figure 2a) consisting of 500 x 500 pm struts in 5 mm length and with a total scaffold height of 4 mm to mimic the macromorphology of bone tissue. Figure 2b depicts the optical microscopy images of the equilibrium swollen (in PBS) hydrogel scaffolds prepared according to example 8. The obtained strut diameter after the removal of the unreacted moieties and obtaining equilibrium swelling in PBS corresponds to 369 ± 55 pm, which implies a CAD / CAM mimicry of 74 ± 1 1 %, while the pore size corresponds to 582 ± 43 pm, implying 1 16 ± 9 % CAD / CAM mimicry. Figure 2c shows a SEM micrograph of the 3D printed hydrogel scaffolds after drying.

[0357] Example 9

[0358] To determine the effect of DFEA or TFEA monomer on the crosslinking kinetics, the DFEA or TFEA monomer was added to a solution of gel-MA-100 in DMSO (10 w / v %), in various molar concentrations and the resulting solutions were subjected to in situ rheological evaluation. The results were compared to a solution of gel-MA-100 in DMSO (10 w / v %) without addition of DFEA (Fig. 3a), or a solution of gel-MA-100 in DMSO or water (10 w / v %), both without addition of TFEA (Fig. 3b).

[0359] From the results in situ rheology analysis depicted in Figures 3a and 3b, it can be seen that the presence of the DFEA or TFEA monomer substantially accelerates the crosslinking kinetics of

[0360] SUBSTITUTE SHEET (RULE 26) the modified gelatins. Without willing to be bound be any theory, this acceleration may be caused by the higher content of mobile cross-linkable acrylamide groups in the reaction mixture. The acceleration of the crosslinking kinetics provides an improvement in terms of CAD-CAM mimicry in case of light-based 3D-printing.

[0361] The detection limits of the cross-linked polymer materials prepared in example 5, having a different content of DFEA, were assessed using19F MRI in vitro io determine the detection limits of the depot which could potentially be still detected in vivo (i.e. , within a 20 min acquisition time).

[0362] The results of the in vitro19F MRI are visualized in Figure 4, by providing19F MRI images of the polymer materials of different masses in Eppendorf vials, from which it can be seen that all polymers could be visualized in depots below 32 mg (corresponding to 32 pL of polymer material) of the swollen polymer. Such low limits of detection ensure an excellent detectability of depots even in later degradation phases and may enable monitoring of polymer material degradation at the site of implantation. Figures 5a, 5b, 5c show the signal to noise (S / N) ratio evaluated from the19F MRI measurement. In general, it can be seen in figures 5a, 5b, 5c, that with the increasing DFEA content, the S / N ratio increases.

[0363] The detection limits of polymer materials containing TFEA, one containing gel-MA-100 prepared according to example 6b (TF1 ), and two containing alg-MA prepared according to example 7 (ATF1 -ATF2), respectively, were studied. It was found that all polymers could be visualized in depots below 40 mg of the swollen polymer.

[0364] Figures 6a, 6b, 6c show the cell seeding study exploiting adipose tissue-derived stem cells. The results show that the materials are cell-interactive and thus, the addition of DFEA monomer into the crosslinked gelatin polymer material network doesn’t affect the cell interactive properties.

[0365] Figure 7 shows the results of the cell viability analysis of selected crosslinked polymer materials with DFEA (G2, G3, G6, G7, G10, G1 1 ) compared to a control. In contrast to gel-MA based polymer materials crosslinked with HEMA, as reported by Dragusin et al., it was surprisingly found that the cell viability of gel-MA based crosslinked polymer materials crosslinked with DFEA did not decrease relative to the control.

[0366] SUBSTITUTE SHEET (RULE 26)

Claims

CLAIMS1 . A crosslinked polymer material detectable by magnetic resonance imaging (MRI), in particular detectable by19F MRI, comprising: a biocompatible polymeric backbone comprising primary groups; imaging groups comprising one or more fluorine atoms; and linker groups providing a linker between the primary groups and the imaging groups; wherein at least part of the primary groups are covalently bonded to the linker groups; wherein the imaging groups are covalently bonded to each other and / or to one or more of the linker groups; wherein crosslinks connecting two or more of the linker groups comprise one or more imaging groups; and wherein the imaging groups are represented by formula (II)whereinRi is selected from -H, and -CH3;X is selected from -O-, and -NR3-;R2 is selected from -Ci ealkyl, and -Cs ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom; andR3 is selected from -H, and -Ci salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; or when X is -NR3-, then R2 and R3 together with the N atom to which they are attached form a 5-6 membered heterocycle; wherein said heterocycle is substituted with at least one -F atom.

2. The crosslinked polymer material as claimed in claim 1 , wherein the biocompatible polymer backbone is a biopolymer selected from polypeptides, proteins, polysaccharides, and polynucleic acids; in particular a biopolymer selected from gelatins, collagens, alginates, dextrans, agarose, glycosaminoglycans, chitosans and carrageenans.

3. The crosslinked polymer material as claimed in claim 1 or 2, wherein the primary groupscomprise amine groups and / or hydroxyl groups and / or carboxylic acid groups.

4. The crosslinked polymer material as claimed in any one of claims 1 to 3, wherein the linker groups comprise a propanoic or a 2-methylpropanoic moiety.

5. The crosslinked polymer material as claimed in any one of claims 1 to 4, wherein the imaging groups are represented by formula (Ila)whereinRi is selected from -H, and -CH3;R2 is selected from -Ci ealkyl, and -Cs ecycloalkyl; wherein said -Ci ealkyl, and -Cs-ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom.

6. The crosslinked polymer material as claimed in any one of claims 1 to 4, wherein the imaging groups are represented by formula (lib)whereinR1 is selected from -H, and -CH3;R2 is selected from -Ci ealkyl, and -Cs ecycloalkyl; wherein said -Ci ealkyl, and -Cs-ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom; andR3 is selected from -H, and -Ci salkyl; wherein said -Ci salkyl is optionally substituted withone or more substituents independently selected from -F, and -OH; or R2 and R3 together with the N atom to which they are attached form a 5-6 membered heterocycle; wherein said heterocycle is substituted with at least one -F atom.

8. A method to prepare the crosslinked polymer material as claimed in any one of claims 1 to 7; comprising the steps of: a) providing a biocompatible polymeric backbone comprising primary groups; b) reacting at least part of the primary groups with a pre-linker group introducing agent to covalently bind pre-linker groups comprising at least one C-C double bond with the primary groups; and c) crosslinking at least part of the pre-linker groups with monomers comprising at least one C-C double bond by free radical chain growth polymerization, wherein at least part of the monomers are imaging monomers comprising one or more fluorine atoms; wherein the imaging monomers are represented by formula (IV)whereinRi is selected from -H, and -CH3;X is selected from -O-, and -NR3-;R2 is selected from -Ci ealkyl, and -Cs-ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom; andR3 is selected from -H, and -Ci salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; or when X is -NR3-, then R2 and R3 together with the N atom to which they are attached form a 5-6 membered heterocycle; wherein said heterocycle is substituted with at least one -F atom.

9. The method as claimed in claim 8, wherein the primary groups comprise amine groups and / or hydroxyl groups and / or carboxylic acid groups.

10. The method as claimed in claim 8 or 9, wherein the pre-linker group introducing agent in step b) is an acrylating agent, a methacrylating agent, or any combination thereof, thereby providing pre-linker groups comprising acrylic moieties, methacrylic moieties, or any combination thereof.1 1 . The method as claimed in any one of claims 8 to 10, wherein the imaging monomers are represented by formula (IVa)whereinR1 is selected from -H, and -CH3;Rz is selected from -Ci ealkyl, and -Cs ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom.

12. The method as claimed in any one of claims 8 to 10, wherein the imaging monomers are represented by formula (IVb)whereinR1 is selected from -H, and -CH3;R2 is selected from -Ci ealkyl, and -Cs ecycloalkyl; wherein said -Ci ealkyl, and -Cs ecycloalkyl is substituted with one or more substituents independently selected from -F, and -OH; wherein R2 comprises at least one -F atom; andR3 is selected from -H, and -Ci salkyl; wherein said -Ci salkyl is optionally substituted with one or more substituents independently selected from -F, and -OH; or R2 and R3 together with the N atom to which they are attached form a 5-6 membered heterocycle; wherein said heterocycle is substituted with at least one -F atom.

13. The crosslinked polymer material as claimed in any one of claims 1 to 7, or as prepared according to the method as claimed in any one of claims 8 to 12, for use in human and / or veterinary medicine.

14. The crosslinked polymer material as claimed in any one of claims 1 to 7, or as prepared according to the method as claimed in any one of claims 8 to 12, for use in tissue engineering or biofabrication.

15. A medical device comprising the crosslinked polymer material as claimed in any one of claims 1 to 7, or as prepared according to the method as claimed in any one of claims 8 to 12.

Images