EP4728008A1

EP4728008A1 - Photo-crosslinkable and 3d printable amphibian collagen derivatives

Info

Publication number: EP4728008A1
Application number: EP24823814.9A
Authority: EP
Inventors: Chor Yong TAY; Jun Kit WANG; Yee Hong Kenrick KOH
Original assignee: Nanyang Technological University
Current assignee: Nanyang Technological University
Priority date: 2023-06-13
Filing date: 2024-06-13
Publication date: 2026-04-22
Also published as: WO2024258349A1; CN121263483A

Abstract

Disclosed herein is a hydrogel precursor formulation that comprises one or both of a methacrylated bullfrog collagen (ColMA) and a methacrylated gelatin (GelMA), where the gelatin is obtained from bullfrog collagen, and a photoinitiator Also disclosed herein is a bioink comprising the aforementioned hydrogel precursor formulation. Further disclosed herein is a kit of parts comprising a ColMA, a GelMA, and a photoinitiator.

Description

PHOTO-CROSSLINKABLE AND 3D PRINTABLE AMPHIBIAN COLLAGEN DERIVATIVES

Field of Invention

The current invention relates to a hydrogel precursor formulation, a methacrylated bullfrog collagen (ColMA) and/or methacrylated bullfrog gelatin (GelMA) suitable for use in the formation of a hydrogel, a bioink comprising the hydrogel precursor formulation, a kit of parts comprising the methacrylated bullfrog collagen, a hydrogel formed by the crosslinking of the methacrylated bullfrog collagen (ColMA) and/or methacrylated bullfrog gelatin (GelMA), use of the hydrogel precursor formulation to encapsulate cells, and a method of encapsulating cells comprising providing a mixture of the hydrogel precursor formulation and cells.

Background

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Gelatin methacryloyl (GelMA) hydrogels have been extensively used for biomedical applications such as scaffolding materials or drug carriers, in lieu to their excellent photo- responsive characteristic that enables the in situ tunability of physical properties to support tissue regeneration or to tailor the drug release behavior (Yue, K. et al., Biomaterials, 2015, 73, 254; Kopecek, J. and Yang, J., Polymer International, 2007, 56, 1078). In addition, GelMA hydrogels are highly biocompatible, owing to their native fibrillar network structure enriched with cell-attachment motifs that support cell attachment and proliferation, mimicking the extracellular matrix (ECM). As a photocurable biomaterial, GelMA has also been widely used as a bioink in 3D bioprinting process to fabricate artificial tissues capable of replacing tissues and organs (Choi, E. et al., Regenerative Biomaterials, 2021 , 8, rbab001).

GelMA was first introduced by Van Den Bulcke et al. in year 2000, in which the methacrylic anhydride (MAA) monomers were reacted with the lysine and hydroxylysine groups of the gelatin at 50 °C (Van Den Bulcke et al., Biomacrocolecules, 2000, 1, 31 ; Shirahama, H. et al., Scientific Reports, 2016, 6, 31036). However, as gelatin is the thermally denatured or disintegrated product of collagen, it does not possess the triple-helix organization of collagen, which is a crucial cell-material interaction motif (Gorgieva, S. and Kokoi, V., Biomaterials Applications for Nanomedicine, 2011 , 2, 17). Furthermore, gelatin exhibits poor structural stability due to its high susceptibility to degradation by proteases found in the human body (Suvarnapathaki, S. et al., RSC Advances, 2019, 9, 13016). As a bioink, the low viscosity of GelMA results in poor resolution in the printed structure coupled with fast gravity-driven sedimentation of cells, particularly with the extrusion-based bioprinters (Pepelanova, I. et al., Bioengineering, 2018, 5, 55; Chen, N. et al., ACS Applied Materials & Interfaces, 2019, 11, 30585). On the other hand, collagen is resistant to most proteases, requiring specific collagenases for enzymatic degradation, and in addition exhibits high viscosity at high concentrations. Thus, photo-responsive collagen materials can overcome conventional disadvantages attributed to photo-responsive gelatin.

Various studies have attempted the development of photo-responsive collagen, such as ultraviolet (UV) crosslinking in the presence of flavin mononucleotide (Ibusuki, S. et al., Tissue Engineering, 2007, 13, 1995), chemically modifying collagen with a photosensitive cinnamate moiety (Dong, C.-M. et al., Biomaterials, 2005, 26, 4041), and chemically modifying collagen with acrylate/methacrylate functional groups prior to photoinitiator-activated crosslinking (US Patent No. US8658711 B2; Poshusta, A.K. and Anseth, K.S., Cells Tissues Organs, 2001 , 169, 272). However, the fabricated photo-responsive collagen gels host a repertoire of issues. For instance, photocrosslinking of the collagen with the flavin mononucleotide resulted in a minimal improvement in terms of the hydrogel’s mechanical properties, while the wavelength used to crosslink the photosensitive cinnamate moiety is cytotoxic in nature (Gaudet, I D. and Shreiber, D.I., Biointerphases, 2012, 7, 25).

Similarly, it is challenging to modify collagen materials via MAA for development of collagenmethacryloyl (ColMA), because the modification chemistry is highly pH- and temperaturedependent. In general, for the MAA modification, a high pH is required to keep the free amino acid groups neutral in order for the MAA reaction to occur. These dependencies present significant problems. Collagen is commonly extracted from the various tissue samples by two main treatments involving either acids or alkalis. Although alkali treatment is a good method to isolate collagen from aged samples, these alkali-treated collagen loses its ability to form fibrils at physiological conditions (Hattori, S. et al., The Journal of Biochemistry, 1999, 725, 676). Hence, acid solubilisation method has been widely used instead. However, native mammalian collagen can only be dissolved under acidic conditions (Wang J.K. et al., Acta Biomaterialia, 2017, 63, 246). Acid solubilised collagen is almost insoluble in alkaline solutions (Wang J.K. et al., Acta Biomaterialia, 2017, 63, 246; Liu, D. et al., Food Chemistry, 2015, 772, 836), where under high pH the collagen tends to precipitate or aggregate. Due to this poor solubility of collagen at high pH, the modification of collagen is limited, particularly for the methacrylation modification where a high pH is required to keep the free amino groups of lysine neutral for the interaction with MAA molecules (Shirahama, H. et al., Scientific Reports, 2016, 6, 31036). Similarly, MAA modification reactions are conducted in the temperature range of 40 - 60 °C, which exceeds the denaturation temperature of collagen. Hence, there remains a definitive research gap for ColMA fabrication due to the lack of collagen sources that permits high degrees of MAA modification.

Summary of Invention

It is surprising that bullfrog collagen with a unique nanofibril nature was able to retain its solubility even at a high pH, prompting new avenues for the synthesis of photo-sensitive methacrylated collagen.

Here, we develop a sustainably sourced (food processing waste) ColMA hydrogel as a cell encapsulant scaffold. The said invention is fabricated using bullfrog (Rana catesbeiana) skin- derived collagen (BFCol) modified with MAA as illustrated in FIG. 1. In addition, a complete MAA modification reaction was achieved under low reaction temperatures at extended duration, without sacrificing its unique triple helical structure. With the alternatively-sourced and newly synthesized ColMA precursor, a 3D hydrogel scaffold was successfully fabricated via lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP; photoinitiator)-activated crosslinking. At the same time, to the best of our knowledge, the GelMA forms of bullfrog skin have yet to be explored. The current invention also explores the modification of bullfrog skin- derived gelatin (BFGel) with MAA to form GelMA. To be comprehensive, both bullfrog ColMA and GelMA (both Type A and Type B, corresponding to acidic/alkali synthesis respectively) were collectively synthesized, and their physicochemical properties were evaluated. To our knowledge, our studies are also the first to demonstrate the potential of BFCol and BFGel for 3D hydrogel applications. The successful development of the said hydrogels with unique properties could potentially lead to the development of hydrogel arrays for drug screening, 3D cell culture, and 3D bioprinting.

Aspects and embodiments of the current invention will now be discussed by reference to the following numbered clauses.

1. A hydrogel precursor formulation comprising:

(a) one or both of a methacrylated bullfrog collagen (ColMA) and a methacrylated gelatin (GelMA), where the gelatin is obtained from bullfrog collagen; and

(b) a photoinitiator. 2. The hydrogel precursor formulation according to Clause 1, wherein both ColMA and GelMA are present.

3. The hydrogel precursor formulation according to Clause 2, wherein a weight to weight ratio of ColMA to GelMa is from 1 :7.5 to 1 :90, such as from 1 :10 to 1 :30, such as from 1 :10 to 1:20.

4. The hydrogel precursor formulation according to any one of the preceding clauses, wherein the photoinitiator is selected from one or more of the group consisting of irgacure, and more particularly, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 2-hydroxy-1-[4-(2- hydroxyethoxy)phenyl]-2-methyl-1-propanone (lrgacure-2959), 2,2’-Azobis[2-Methyl-N-(2- hydroxyethyl) propionamide] (VA-086), and eosin Y (EY), optionally wherein the photoinitiator is LAP.

5. The hydrogel precursor formulation according to any one of the preceding clauses, wherein the ColMA has a triple helical structure of collagen that is retained and is not denatured.

6. The hydrogel precursor formulation according to any one of the preceding clauses, wherein the ColMA has a free amine group amount of from 15 to 50%, such as from 20 to 45%, such as from 25 to 35%.

7. The hydrogel precursor formulation according to any one of the preceding clauses, wherein the ColMA, when formed into a hydrogel, has a storage modulus of from 1 to 4 kPa, such as from 2 to 3 kPa.

8. The hydrogel precursor formulation according to any one of the preceding clauses, wherein the ColMA, when formed into a hydrogel, undergoes a shrinkage of from 15 to 30%, such as from 18 to 25%, such as about 20%.

9. The hydrogel precursor formulation according to any one of the preceding clauses, wherein the GelMA has one or more of the following properties when present:

(ia) a free amine group amount of from 25 to 50%, such as from 25.5 to 45%;

(ib) a histidine count of one per 1 ,000 residues; and

(ic) a solubility limit of about 60% w/v in water at room temperature. 10. The hydrogel precursor formulation according to any one of the preceding clauses, wherein the GelMA, when formed into a hydrogel, has one or more of the following properties:

(aa) a storage modulus of from 15 to 30 kPa, such as from 18 to 28 kPa;

(ab) a dimension change of from -5% to 5%, such as from -3% to 2%.

11 The hydrogel precursor formulation according to any one of the preceding clauses, wherein a weight to weight ratio of the photoinitiator to:

(ba) ColMA is from 1 :2 to 1:10, such as from 1:5 to 1 :7.5; and/or

(be) GelMA is from 1:50 to 1 :300, such as from 1 :50 to 1 :200, or from 1:75 to 1 :150.

12. A methacrylated bullfrog collagen (ColMA) suitable for use in the formation of a hydrogel.

13. The ColMa according to Clause 12, wherein the ColMA has one or more of the following properties:

(ca) the ColMA has a triple helical structure of collagen that is retained and is not denatured;

(cb) the ColMA has a free amine group amount of from 15 to 50%, such as from 20 to 45%, such as from 25 to 35%;

(cc) the ColMA, when formed into a hydrogel, has a storage modulus of from 1 to 4 kPa, such as from 2 to 3 kPa;

(cd) the ColMA, when formed into a hydrogel, undergoes a shrinkage of from 15 to 30%, such as from 18 to 25%, such as about 20%.

14. A bioink comprising a hydrogel precursor formulation according to any one of Clauses 1 to 11.

15. The bioink according to Clause 14, further comprising an aqueous solvent.

16. A kit of parts comprising:

(i) a methacrylated bullfrog collagen (ColMA) in an acidic aqueous medium;

(ii) a methacrylated gelatin (GelMA) in an aqueous medium; and

(iii) a photoinitiator, optionally wherein the photoinitiator is selected from one or more of the group consisting of irgacure, and more particularly, lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1- propanone (lrgacure-2959), 2,2’-Azobis[2-Methyl-N-(2-hydroxyethyl) propionamide] (VA-086), and eosin Y (EY), further optionally wherein the photoinitiator is LAP. 17. The kit of parts according to Clause 16, wherein one or more of the following apply:

(da) the ColMA has a triple helical structure of collagen that is retained and is not denatured;

(db) the ColMA has a free amine group amount of from 15 to 50%, such as from 20 to 45%, such as from 25 to 35%;

(de) the ColMA, when formed into a hydrogel, has a storage modulus of from 1 to 4 kPa, such as from 2 to 3 kPa;

(dd) the ColMA, when formed into a hydrogel, undergoes a shrinkage of from 15 to 30%, such as from 18 to 25%, such as about 20%;

(de) the GelMA has a free amine group amount of from 25 to 50%, such as from 25.5 to 45%;

(df) the GelMA, when formed into a hydrogel, has a storage modulus of from 15 to 30 kPa, such as from 18 to 28 kPa;

(dg) the GelMA, when formed into a hydrogel, has a dimension change of from -5% to 5%, such as from -3% to 2%;

(dh) the GelMA has a histidine count of one per 1 ,000 residues; and

(di) the GelMA has a solubility limit of about 60% w/v in water at room temperature.

18. A kit of parts comprising:

(ai) a methacrylated bullfrog collagen (ColMA) in an acidic aqueous medium; and

(aii) a photoinitiator, optionally wherein the photoinitiator is selected from one or more of the group consisting of irgacure, and more particularly, lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1- propanone (lrgacure-2959), 2,2’-Azobis[2-Methyl-N-(2-hydroxyethyl) propionamide] (VA-086), and eosin Y (EY), further optionally wherein the photoinitiator is LAP.

19. The kit of parts according to Clause 18, wherein one or more of the following apply:

(ea) the ColMA has a triple helical structure of collagen that is retained and is not denatured;

(eb) the ColMA has a free amine group amount of from 15 to 50%, such as from 20 to 45%, such as from 25 to 35%;

(ec) the ColMA, when formed into a hydrogel, has a storage modulus of from 1 to 4 kPa, such as from 2 to 3 kPa;

(ed) the ColMA, when formed into a hydrogel, undergoes a shrinkage of from 15 to 30%, such as from 18 to 25%, such as about 20%.

20. A hydrogel formed by the crosslinking of a methacrylated bullfrog collagen (ColMA). 21. The hydrogel according to Clause 20, wherein the hydrogel has one or both of the following properties:

(fa) a storage modulus of from 1 to 4 kPa, such as from 2 to 3 kPa; and

(fb) a shrinkage of from 15 to 30%, such as from 18 to 25%, such as about 20%, optionally wherein the photoinitiator is selected from one or more of the group consisting of irgacure, and more particularly lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 2-hydroxy-1-[4-(2- hydroxyethoxy)phenyl]-2-methyl-1-propanone (lrgacure-2959), 2,2’-Azobis[2-Methyl-N-(2- hydroxyethyl) propionamide] (VA-086), and eosin Y (EY), further optionally wherein the photoinitiator is LAP.

22. Use of a hydrogel precursor formulation as described in any one of Clauses 1 to 11 to encapsulate cells, optionally wherein the cells are living and remain in a viable state after encapsulation.

23. A method of encapsulating cells, the method comprising:

(ga) providing a mixture of a hydrogel precursor formulation according to any one of Clauses 1 to 11 and cells; and

(gb) subjecting the mixture to light in order to cause the hydrogel precursor formulation to form a hydrogel and encapsulate the cells, optionally wherein the cells are living and remain in a viable state after encapsulation.

24. A methacrylated gelatin (GelMA) suitable for use in the formation of a hydrogel, wherein the gelatin is obtained from bullfrog collagen.

25. The GelMA according to Clause 24, wherein the GelMA has one or more of the following properties:

(ga) a free amine group amount of from 25 to 50%, such as from 25.5 to 45%;

(gb) a histidine count of one per 1 ,000 residues; and

(gc) a solubility limit of about 60% w/v in water at room temperature.

26. The GelMA according to Clause 24 or Clause 25, wherein the GelMA, when formed into a hydrogel, has one or more of the following properties:

(aa) a storage modulus of from 15 to 30 kPa, such as from 18 to 28 kPa; and

(ab) a dimension change of from -5% to 5%, such as from -3% to 2%.

27. A kit of parts comprising: (iia) a methacrylated gelatin (GelMA) in an aqueous medium, wherein the gelatin is obtained from bullfrog collagen; and

(ib) a photoinitiator, optionally wherein the photoinitiator is selected from one or more of the group consisting of irgacure, and more particularly, lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1- propanone (lrgacure-2959), 2,2’-Azobis[2-Methyl-N-(2-hydroxyethyl) propionamide] (VA-086), and eosin Y (EY), further optionally wherein the photoinitiator is LAP.

28. The kit of parts according to Clause 27, wherein one or more of the following apply:

(aaa) the GelMA has a free amine group amount of from 25 to 50%, such as from 25.5 to 45%;

(aab) the GelMA, when formed into a hydrogel, has a storage modulus of from 15 to 30 kPa, such as from 18 to 28 kPa;

(aac) the GelMA, when formed into a hydrogel, has a dimension change of from -5% to 5%, such as from -3% to 2%;

(aad) the GelMA has a histidine count of one per 1 ,000 residues; and

(aae) the GelMA has a solubility limit of about 60% w/v in water at room temperature.

29. A hydrogel formed by the crosslinking of a methacrylated bullfrog collagen (ColMA) with a methacrylated gelatin (GelMA), wherein the gelatin is obtained from bullfrog collagen.

30. A hydrogel formed by the crosslinking of a methacrylated gelatin (GelMA), wherein the gelatin is obtained from bullfrog collagen.

Drawings

FIG. 1 depicts the schematic illustration of collagen-methacryloyl (ColMA) or gelatin- methacryloyl synthesis using bullfrog skins (i . e. , food waste) as a sustainable source of protein. The bullfrog skins were kindly contributed by Jurong Frog Farm, Singapore.

FIG. 2 depicts the characterisation of ColMA precursors: (a) 2,4,6-trinitrobenzene sulfonic acid (TNBS) profile and (b) circular dichroism (CD) analysis.

FIG. 3 depicts the characterization of ColMA hydrogels: (a) representative digital pictures of the fabrication of a ColMA hydrogel. The first image on the far left of (a) shows a ColMA precursor dissolved in 0.5 M acetic acid in the presence of phenol red (the sample was yellow in colour), the central image of (a) shows the ColMA precursor after neutralization (the sample was red in colour), and image on the far right of (a) shows the ColMA hydrogel sample after exposure to UV (the sample was colourless); (b) storage modulus; (c) gel shrinkage; and (d) degradation profile.

FIG. 4 depicts the characterisation of BFGel: (a) digital picture, (b) attenuated total reflectance Fourier transform infrared (ATR-FTIR) profile, (c) CD analysis, and (d) TNBS assay.

FIG. 5 depicts the characterization of GelMA precursors: TNBS profile.

FIG. 6 depicts the characterization of GelMA hydrogels: (a) representative digital pictures of the fabrication of a GelMA hydrogel. The first image on the far left of (a) shows a GelMA precursor dissolved in DI water in the presence of phenol red (the sample was yellow in colour), the central image of (a) shows the GelMA precursor after neutralization (the sample was red in colour), and image on the far right of (a) shows the GelMA hydrogel sample after exposure to UV (the sample was slight yellow in colour due to high concentration of GelMA); (b) storage modulus; (c) gel shrinkage; and (d) degradation profile.

FIG. 7 depicts the LIVE/DEAD fluorescence images of human keratinocytes (HaCaT) cells encapsulated within the ColMA and GelMA hydrogels (Scale bar = 100 pm).

FIG. 8 depicts the 3D bioprinting of ColMA-GelMA hybrid system using (a) Lumen X+ lightbased bioprinter (adapted from https://www.cellink.com/bioprintinQ/lumen-x/); and (b) the constructed hybrid hydrogels.

FIG. 9 depicts the rheological characterisation of the hybrid ColMA-GelMA hydrogels.

FIG. 10 depicts the LIVE/DEAD staining of the 3D encapsulated HDF cells.

Description

The scarcity of studies on ColMA could be attributed to the lack of the sources of collagen that can tolerate the high pH modification. The alternatively-sourced bullfrog collagen, valorized from bullfrog skin discards, was able to directly address the aforementioned issue. The methacrylation modification enhanced the functionality of the collagen gel without sacrificing the triple helical structure of the ColMA hydrogels, thereby surprisingly allowing the application of bullfrog collagen (and gelatin) to form hydrogels (and hydrogel precursors) with particularly useful properties. Thus, in a first aspect of the invention, there is provided a hydrogel precursor formulation comprising:

(b) a photoinitiator.

Bullfrog (BF) collagen is surprisingly resistant to high pH values, thereby allowing it to form useful forms of methacrylated bullfrog collagen (ColMA), which may then be used in the formation of hydrogels and therefore hydrogel precursor formulations. In particular, it is surprising that the triple helical structure of the collagen is preserved during the methacrylation process. Similarly, it has been surprisingly found that bullfrog-derived gelatin is different to mammalian-derived gelatin in its amino acid make-up (see Table A below), as well as the physical properties of methacrylated gelatins (GelMAs) derived from said bullfrog gelatin. In particular, bullfrog GelMA boasts a higher solubility limit, reaching approximately 60% w/v, in contrast to other sources like commercial GelMA from porcine, which typically gelated around 15% w/v at room temperature. Given the same degree of modification, bullfrog GelMA enables a broader spectrum of photocrosslinking formulations and facilitates easier handling.

The distinctive variations in composition between bullfrog-derived GelMA and commercially available GelMA make it easily distinguishable through amino acid profiling.

Table A

As will be appreciated, the hydrogel precursor formulation may contain one or both of GelMA and ColMA. In particular embodiments of the invention that may be mentioned herein, both ColMA and GelMA may be present.

In embodiments of the invention where both ColMA and GelMA are present, any suitable weight to weight ratio of ColMA to GelMa may be used. For example, the weight to weight ratio of ColMA to GelMa may be from 1 :7.5 to 1 :90, such as from 1 :10 to 1 :30, such as from 1:10 to 1 :20.

In some examples of the invention that may be mentioned herein, the following weight to weight ratios of ColMA to GelMa may be used, as shown in Table B.

Table B

As noted hereinbefore, the hydrogel precursor formulation contains a photoinitiator. Any suitable photoinitiator material that is compatible with the methacrylate functional groups may be used herein. For example, the photoinitiator may be selected from one or more of the group consisting of irgacure, and more particularly, lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1- propanone (lrgacure-2959), 2,2’-Azobis[2-Methyl-N-(2-hydroxyethyl) propionamide] (VA-086), and eosin Y (EY), optionally wherein the photoinitiator is LAP. As noted hereinbefore, the ColMA may retain its triple helical structure, even after extraction and methacrylation. Thus, in further embodiments of the invention that may be mentioned herein, the ColMA may have a triple helical structure of collagen that is retained and is not denatured.

While not essential, it is believed that better mechanical and physical properties are retained in a ColMA that retains at least some free amine groups. Thus, in an embodiment of the invention, the ColMA may have a free amine group amount of from 15 to 50%, such as from 20 to 45%, such as from 25 to 35%.

For the avoidance of doubt, reference to the percentage of free amine groups present in the ColMA or GelMA disclosed herein refers to the percentage of free amines in a ColMA or GelMA sample relative to the quantified total number of amines present in the sample. The number of free amines in a sample can be quantified using methods known in the art, for example by using a TNBS assay. The total number of amines present in the ColMA or GelMA sample may be quantified by determining the number of free amines present in a corresponding collagen or gelatin sample before modification with MAA. In certain exemplary embodiments, the number of free amines in a given sample is quantified using a TNBS assay.

The ColMA may have any suitable storage modulus when formed into a hydrogel, but in particular embodiments that may be mentioned herein, the ColMA, when formed into a hydrogel, has a storage modulus of from 1 to 4 kPa, such as from 2 to 3 kPa.

The ColMA may have any suitable shrinkage when formed into a hydrogel, but in particular embodiments that may be mentioned herein, the ColMA, when formed into a hydrogel, undergoes a shrinkage of from 15 to 30%, such as from 18 to 25%, such as about 20%.

For the avoidance of doubt, the term “shrinkage” with reference to a hydrogel disclosed herein, and any reference to the percentage of shrinkage of a hydrogel disclosed herein, refers to the reduction in size of the hydrogel following exposure to a physiological buffer for a given time period relative to the size of the hydrogel before exposure to the physiological buffer for the given time period. For example, in certain embodiments, the hydrogel may undergo a shrinkage of from 15 to 30% (e.g. from 18 to 25%, such as about 20%) when exposed to a phosphate buffered saline (PBS) for 1 day at room temperature relative to the size of the hydrogel before exposure to the PBS for 1 day at room temperature. Similarly, the term “dimension change” with reference to a hydrogel disclosed herein, and any reference to the percentage of dimension change of a hydrogel disclosed herein, refers to the change in a dimension (e g. height, width and/or length) of the hydrogel following exposure to a physiological buffer for a given time period relative to the dimension of the hydrogel before exposure to the physiological buffer for the given time period. For example, in certain embodiments, a dimension of the hydrogel may change by from -5% to 5% (e.g. from -3% to 2%) when exposed to a phosphate buffered saline (PBS) for 1 day at room temperature relative to the dimension of the hydrogel before exposure to the PBS for 1 day at room temperature.

It will be appreciated that the storage modulus and shrinkage are measured using a hydrogel (formed as described in the examples section) that contains only ColMA and does not also contain GelMA.

The GelMA may have one or more of the following properties:

(ia) a free amine group amount of from 25 to 50%, such as from 25.5 to 45%;

(ib) a histidine count of one per 1 ,000 residues; and

(ic) a solubility limit of about 60% w/v in water (e.g. deionised water) at room temperature (i.e. about 22 °C to about 25 °C).

The GelMA may have any suitable storage modulus when formed into a hydrogel, but in particular embodiments that may be mentioned herein, the GelMA, when formed into a hydrogel, may have a storage modulus of from 15 to 30 kPa, such as from 18 to 28 kPa.

The GelMA may have any suitable dimension change when formed into a hydrogel, but in particular embodiments that may be mentioned herein, the GelMA, when formed into a hydrogel, may have a dimension change of from -5% to 5%, such as from -3% to 2%.

It will be appreciated that the storage modulus and shrinkage are measured using a hydrogel (formed as described in the examples section) that contains only GelMA and does not also contain ColMA.

The photoinitiator may be present in any suitable amount in the hydrogel precursor formulation. For example, the hydrogel precursor formulation may be one in which a weight to weight ratio of the photoinitiator to:

(ba) ColMA is from 1 :2 to 1:10, such as from 1:5 to 1 :7.5; and/or

(be) GelMA is from 1:50 to 1 :300, such as from 1 :50 to 1 :200, or from 1:75 to 1 :150. In a further aspect of the invention, there is provided a methacrylated bullfrog collagen (ColMA) suitable for use in the formation of a hydrogel. As will be appreciated, the properties of the ColMA described in this aspect are as described hereinbefore for the ColMA as part of a hydrogel precursor formulation and so this discussion is omitted here for brevity.

In a further aspect of the invention, there is provided a methacrylated gelatin (GelMA) suitable for use in the formation of a hydrogel, wherein the gelatin is obtained from bullfrog collagen. As will be appreciated, the properties of the GelMA described in this aspect are as described hereinbefore for the GelMA as part of a hydrogel precursor formulation and so this discussion is omitted here for brevity.

In yet a further aspect of the invention, there is provided a bioink comprising a hydrogel precursor formulation as described hereinbefore. In embodiments of this aspect, the bioink may further comprise an aqueous solvent.

In a further aspect of the invention, there is provided a kit of parts comprising:

(i) a methacrylated bullfrog collagen (ColMA) in an acidic aqueous medium;

(ii) a methacrylated gelatin (GelMA) in an aqueous medium; and

(iii) a photoinitiator, optionally wherein the photoinitiator is selected from one or more of the group consisting of irgacure and, more particularly, lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1- propanone (lrgacure-2959), 2,2’-Azobis[2-Methyl-N-(2-hydroxyethyl) propionamide] (VA-086), and eosin Y (EY), further optionally wherein the photoinitiator is LAP.

In embodiments of the invention, the kit of parts of this aspect may have one or more of the following features:

(de) the GelMA has a free amine group amount of from 25 to 50%, such as from 25.5 to 45%; (df) the GelMA, when formed into a hydrogel, has a storage modulus of from 15 to 30 kPa, such as from 18 to 28 kPa;

(dh) the GelMA has a histidine count of one per 1 ,000 residues; and

(di) the GelMA has a solubility limit of about 60% w/v in water (e.g. deionised water) at room temperature (i.e. about 22 °C to about 25 °C).

In yet a further aspect of the invention, there is provided a kit of parts comprising:

(ai) a methacrylated bullfrog collagen (ColMA) in an acidic aqueous medium; and

(aii) a photoinitiator, optionally wherein the photoinitiator is selected from one or more of the group consisting of irgacure, and more particularly, lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1- propanone (lrgacure-2959), 2,2’-Azobis[2-Methyl-N-(2-hydroxyethyl) propionamide] (VA-086), and eosin Y (EY), further optionally wherein the photoinitiator is LAP. In embodiments of said aspect, one or more of the following may apply:

(iia) a methacrylated gelatin (GelMA) in an aqueous medium, wherein the gelatin is obtained from bullfrog collagen; and

(ib) a photoinitiator, optionally wherein the photoinitiator is selected from one or more of the group consisting of irgacure, and more particularly, lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1- propanone (lrgacure-2959), 2,2’-Azobis[2-Methyl-N-(2-hydroxyethyl) propionamide] (VA-086), and eosin Y (EY), further optionally wherein the photoinitiator is LAP. In embodiments of this aspect, one or more of the following may apply:

(aab) the GelMA, when formed into a hydrogel, has a storage modulus of from 15 to 30 kPa, such as from 18 to 28 kPa; (aac) the GelMA, when formed into a hydrogel, has a dimension change of from -5% to 5%, such as from -3% to 2%;

(aad) the GelMA has a histidine count of one per 1 ,000 residues; and

(aae) the GelMA has a solubility limit of about 60% w/v in water (e.g. deionised water) at room temperature (i.e. about 22 °C to about 25 °C).

In a further aspect of the invention, there is provided a hydrogel formed by the crosslinking of a methacrylated bullfrog collagen (ColMA). The ColMA (and the hydrogel thereof) may have the properties described hereinbefore, which are omitted here for the sake of brevity.

In a further aspect of the invention, there is provided a hydrogel formed by the crosslinking of a methacrylated bullfrog collagen (ColMA) with a methacrylated gelatin (GelMA), wherein the gelatin is obtained from bullfrog collagen. The ColMA and GelMA may have the properties described hereinbefore, which are omitted here for the sake of brevity.

In a further aspect of the invention, there is provided a hydrogel formed by the crosslinking of a methacrylated gelatin (GelMA), wherein the gelatin is obtained from bullfrog collagen. The GelMA (and the hydrogel thereof) may have the properties described hereinbefore, which are omitted here for the sake of brevity.

In a further aspect of the invention, there is provided a use of a hydrogel precursor formulation as described hereinbefore to encapsulate cells, optionally wherein the cells are living and remain in a viable state after encapsulation.

In a further aspect of the invention, there is provided a method of encapsulating cells, the method comprising:

(ga) providing a mixture of a hydrogel precursor formulation as described herein and cells; and

The current invention provides the following advantages and improvements.

1. Synthesized a photo-responsive hydrogel precursor using bullfrog collagen with preserved triple helical structure. Compared to conventional mammalian-based collagen, BFCol does not undergo aggregation easily at high alkaline pH range, thus expanding its versatility for downstream modifications.

2. Synthesized a photo-responsive hydrogel precursor using bullfrog gelatin of Type A and Type B.

3. Demonstrated the functionality of bullfrog-based collagen as photo-responsive materials with distinct physicochemical properties. To our knowledge, our studies are also the first to demonstrate the potential of BFCol and BFGel for 3D hydrogel applications.

4. The successful development of the said hydrogels with unique properties could potentially lead to the development of hydrogel arrays for drug screening, 3D cell culture, and 3D bioprinting.

Collagen is commonly extracted from various tissue samples by two main treatments involving either acids or alkalis. Although alkali treatment is a good method to isolate collagen from aged samples, these alkali-treated collagen loses its ability to form fibrils at physiological conditions. Hence, acid solubilization method has been widely used instead. However, acid solubilized collagen is readily dissolved under acid conditions, while almost insoluble in alkaline solutions. Due to this poor solubility of collagen at high pH, the modification of collagen is limited, particularly for the methacrylation modification where a high pH is required to keep the free amino groups of lysine neutral for the interaction with MAA molecules. Surprisingly, bullfrog collagen with a unique nanofibril nature (e.g. the preparative method described in US Patent Application No. US 2021/0324045A1, which method is incorporated herein by reference) was able to retain its solubility even at a high pH, prompting new avenues for the synthesis of photo-sensitive methacrylated collagen. In addition, the methacrylation modification enhanced the functionality of the collagen gel without sacrificing the triple helical structure of the ColMA hydrogels. As the integrity of the collagen’s triple helical conformation is responsible for its various biological functions such as the regulation of cell differentiation, morphology, migration and adhesion behaviors during tissue repair, preservation of this structure is crucial for its employment into biomedical applications.

Bullfrog ColMA and GelMA were observed to exhibit different physical properties with GelMA showing significantly higher storage modulus compared to ColMA. In general, matrix stiffness plays an important role in modulating various cellular processes, such as cell proliferation, migration, and differentiation. Hence, by combining both ColMA and GelMA at different ratios, hydrogels with a range of stiffness could be produced to influence the cell behaviors. A composite material that can combine simultaneously the advantages of both components to avoid their own defects is desirable. In this case, while collagen is the main component of the extracellular matrix with preserved triple helical structure, it exhibited poor physical properties. On the other hand, gelatin exhibited good physical properties, but it is a non-viscous solution that could have heterogeneous distribution of additive that highly dependent on its mass. A composite of both will make use of the strengths of the components while nullifying their disadvantages. Furthermore, a composite scaffold is more bio-mimicking as compared to a single structure with simple properties, as observed in natural ECM.

Biomaterial selection for 3D bioprinting application is often associated with adjustability of the viscosity, stability/degradability, and rapid crosslinking time either as a scaffold or cell encapsulant material. ColMA is capable of exhibiting high viscosity with preserved triple helical structures, which in tandem enable both cell-material interactions and homogeneous distribution of cells in the precursor solution during the bioprinting process. Despite the advantages of ColMA, the intrinsic self-assembly process of the collagen fibril often associated with high shrinkage of the obtained hydrogels, which could limit its application for tissue engineering. On the other hand, although GelMA has low viscosity, the fabricated hydrogel exhibits high modulus with minimal dimensional changes upon UV-crosslinking. Considering the advantages of ColMA and GelMA, hybrid hydrogels with different concentrations of ColMA and GelMA can be developed as an ideal bioink for 3D bioprinting application. Here, we have designed a new bioink to print a tissue-mimicking hybrid material via Lumen X+ (the first digital light processing printer specifically designed for bioprinting with minimal dimensional changes). This novel photocrosslinkable bioink comprises of pre-dissolved ColMA in 0.5M acetic acid, GelMA in DI water, and a LAP photoinitiator in high concentrations, which were then mixed and neutralized in various ColMA-to-GelMA ratios with a final 0.1 % w/v LAP (i.e., 0.75% w/v ColMA and 7.50% w/v GelMA, and another combination of bioink with 0.50% w/v ColMA and 15.00% w/v GelMA) aiming to produce hydrogels with varying properties upon photocrosslinking. The newly developed bioink enables the printing of complex 3D constructs that are highly compatible with the human body. This could further enhance the scalability of 3D printed tissue manufacturing, with a wide array of applications in regenerative medicine.

Collagen is the most abundant protein in the human body, which is widely used in medical tissue engineering, drug delivery, cosmetics, food, and other fields. Because of its wide applications, it has, in recent years, prompted many researchers to isolate collagen from more sustainable sources, and functionalize the extracted collagen based on application. In medical tissue engineering, additive manufacturing of photosensitive hydrogels has been extensively explored as scaffolding materials for 3D cell culture. Other than the 3D complex structure, photo-responsive hydrogels could also be used for microencapsulation, 3D array for screening, core-shell, or Janus structure with distinct characteristics on each layer, etc. However, there are limited commercially available photosensitive collagen products, and to our best knowledge PhotoCol® (Advanced Biomatrix) is mostly utilized for research. In addition, PhotoCol® is fabricated using a mammalian derived collagen (from bovine hide), which has high monomer content, and the raw material is widely used for valuable leather production. Hence, a sustainable non-mammalian ColMA produced from food waste could represent a cost-effective and “cleaner manufacturing” method to produce high-value biomedical products.

Further aspects and embodiments of the current invention will be described by the following non-limiting examples.

Examples

Materials

Bullfrog skins were collected from Khaiseng Trading & Fish Farm Pte Ltd. Acetic acid, sodium carbonate, sodium bicarbonate, methacrylic anhydride, hydrochloric acid, 2,4,6- trinitrobenzene sulfonic acid, sodium dodecyl sulfate, phenol red, Dulbecco’s Modified Eagle’s Medium high glucose, lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate, fluorescein diacetate and propidium iodide were purchased from Sigma-Aldrich. SnakeSkinTM dialysis tubing (10K MWCO), fetal bovine serum, antibiotic-antimycotic and Hoechst were obtained from Thermo Fisher Scientific. Sodium hydroxide was purchased from Schedelco Pte. Ltd.

Example 1. Fabrication and Characterisation of ColMA Precursors

Methacrylation modification on BFCol

The methacrylated collagen was synthesised according to the protocol established for GelMA synthesis (Shirahama, H. et al., Scientific Reports, 2016, 6, 31036), with slight modifications to buffer strength, reaction time and temperature. Briefly, BFCol was first dissolved in 0.5 M acetic acid at 1% w/v overnight prior to the mixture of carbonate-bicarbonate (CB) buffer at 1:1 volume ratio of collagen and buffer solution. A CB buffer with a buffer strength of 1.5 M (i.e., 7.15 g of sodium carbonate and 0.63 g sodium bicarbonate in 50 mL distilled water) was chosen because it can resist drastic changes in pH level due to the formation of acidic byproducts during the MAA reaction. The mixture was first allowed to stir for 24 h to form a stable pH of 10 - 11 prior to the addition of MAA to the collagen/CB solution at MAA to collagen ratio from 0.1 to 3.0 mL/g where the reaction proceeded for 4 days with pH monitoring throughout the reaction period. At the end of the reaction, the reaction was stopped by neutralizing with 12 M HCI prior to dialysis using 10 K molecular-weight cutoff (MWCO). SnakeSkin™ dialysis tubing against 30% ethanol, 0.1 M acetic acid, and distilled water for 48 h each with a fresh solution changed at 24 h time point before lyophilisation and storage at 4 °C until further usage. All the steps were performed at 4 °C and all the solutions involved were cooled to 4 °C prior to usage.

Determination of Degree of Substitution

To quantify the degree of substitution, 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay was performed as recommended by Thermo Scientific, USA. Briefly, 20-200 pg/mL of the samples were prepared by dissolving in 0.1 M sodium bicarbonate buffer solution (pH 8.5). Thereafter, 0.25 mL of the TNBS solution (0.01% w/v) was added to 0.5 mL of each sample and incubated for 2 h at 37 °C. At the end of the reaction, 0.25 mL of 10% sodium dodecyl sulfate (SDS; dissolved in distilled water) and 0.125 mL of 1 N HCI were added to each sample, in which the absorbance of the solutions was measured using a SpectraMax M2 microplate reader (Molecular Devices, USA) at 335 nm.

Circular Dichroism Analysis

The secondary structure of the samples, such as presence of the triple helix, was evaluated via circular dichroism (CD) using an Aviv Model 420 spectrometer (Aviv Biomedical Inc., USA). Briefly, samples were first dissolved in 0.5 M acetic acid at 10 mg/mL, followed by loading into quartz cuvettes (150 pL, 0.5 mm path length, 106 QS, Hellma, USA). Measurements were the scanned from 250 nm to 190 nm at 1 nm intervals. 0.5 M acetic acid was used as a blank solution for correction.

Results and Discussions

As an amphibian, bullfrogs possess lower body temperatures due to their habitat in comparison to terrestrial mammals, and thus collagen from its skin naturally exhibits lower isoelectric point, a similar phenomenon observed in marine-sourced collagen (Huss, H.H. et al., Fresh Fish : Quality and Quality Changes : A Training Manual; Fao: Rome, 1988; Meyer, M., Biomedical Engineering Online, 2019, 18, 1). The lowered isoelectric point in bullfrog collagen prevents aggregation from the high pH used in the MAA modification when compared to the bovine collagen (Li, R. et al., Biotechnology Reports, 2016, 9, 46). As seen from FIG. 1 , an acidic by-product is produced during the MAA modification reaction. Hence, a strong buffering system, namely carbonate-bicarbonate (CB) buffer was adopted following the previously published protocol of GelMA synthesis (Shirahama, H. et al., Scientific Reports, 2016, 6, 31036; US Patent No. US8658711 B2). The CB buffer has been shown to be more effective than other buffer systems such as phosphate buffered saline (PBS) or 2-(N- morpholino)ethanesulfonic acid (MES) in ensuring complete reaction of MAA with bullfrog collagen, due to the other buffer systems having similar isoelectric point (IEP) to the protein. To counteract the acidic by-products generated by the reaction, the molarity of the CB buffer was increased from the recommended 0.25 M in GelMA system to 0.75 M. Despite the new set of reaction variables, the bullfrog collagen is able to remain in its solubilized form, whereas instant aggregation was observed in the mammalian collagen.

The successful modification of collagen into ColMA was evaluated by TNBS assay. During the modification, the free amine groups of lysine reacted with the MAA molecules covalently to produce methacryloyl moieties functional groups. As such, the degree of modification was confirmed by quantifying the percentage of free amines that remained in the collagen. As shown in FIG. 2a, extracted collagen had been modified into ColMA, as evidenced by decreasing amine functional sites with correspondingly increasing amount of MAA introduced into the system during modification.

In addition, the MAA modification did not cause deterioration to the triple helical structures in collagen, as observed via CD in FIG. 2b. As the integrity of the collagen’s triple helical conformation is responsible for its various biological functions such as the regulation of cell differentiation, morphology, migration and adhesion behaviors during tissue repair, preservation of this structure is crucial for its employment into biomedical applications.

Example 2. Fabrication and Characterisation of ColMA Hydrogels

Lyophilised ColMA used herein were obtained by following the protocol disclosed in Example 1 . The physicochemical properties of the hydrogels were evaluated by measuring the storage modulus, dimensional changes, and degradability.

Fabrication of ColMA Hydrogels

Lyophilised ColMA was dissolved in 0.5 M acetic acid at a concentration of 1.2% w/v. Thereafter, either 10* phenol red solution or 10* serum free medium (SFM) was added into the respective precursors to form a final 1* concentration prior to neutralization using either 22 M sodium hydroxide or 12 M hydrochloric acid. Next, 5 % w/v LAP photoinitiator was prepared and added to the mixture with a final concentration of 0.1 % w/v before the overall volume was adjusted using either 0.5 M acetic acid to form a final ColMA of 1 % w/v. Finally, the mixture was transferred into a desired mold and crosslinked under UV light for 1 min at a distance of 1 cm to produce ColMA hydrogels.

Storage Modulus Determination of ColMA Hydrogels

The rheological properties of the hydrogels were measured with a Physica MCR 501 rheometer to determine the physical property (i.e. , storage modulus; G’) of the samples. The samples were compressed to a normal force of 0.04 N before running the test. The changes in the storage modulus (G’) was determined at 1% strain and a frequency of 1 Hz, in which the storage modulus was quantified.

Dimensional Changes Determination of ColMA Hydrogels

The shrinkage/swelling behavior of the fabricated hydrogels was analysed by comparing the dimensional changes of the hydrogels on Day 0 and Day 1. Briefly, the photographs of the hydrogels on Day 0 and 1 were imaged, in which the dimension of the hydrogels was measured using the Imaged freeware (https://imaqei.nih.gov/ii/).

Degradability Assessment of ColMA Hydrogels

The degradability of the fabricated hydrogels was assessed over 21 days by incubating the samples in PBS solution at 37 °C under a static condition without shaking. On pre-determined time points (i.e., Day 0, 1 , 3, 6, 8, 10, 13, 15, 17, 20, and 21), the solution was collected and replaced by fresh PBS. At the end of 21 days, the degraded products released into the PBS solution was quantified using the Bicinchoninic Acid (BOA) protein assay. In addition, photographs of the hydrogels were recorded on Day 7, 14 and 21 for a visual assessment of the degradation progress.

Results and Discussions

The functionality of the ColMA was confirmed by the photo-responsiveness of the precursor in the presence of LAP photoinitiator upon exposed to UV irradiation (FIG. 3a). Under the UV light, chain growth polymerization reaction occurred by formation of covalent bonds between the methacrylate groups on the lysine side chains, leading to crosslinked networks and thus stable hydrogels.

Rheological measurements of the samples showed the increasing storage modulus with increasing MAA to collagen ratio, attributed to the higher percentage of crosslinked networks within the hydrogels (FIG. 3b). In addition, a reduction in dimensional shrinkage (upon exposure of the hydrogel to 1 x PBS) was also observed with increasing degrees of methacrylation (FIG. 3c), in lieu of the increased physical structure stabilization. In general, a reduction in the hydrogel dimension is commonly accompanied by the self-assembly process into a fibrillar hydrogel. However, in the view of tissue engineering application, a minimal change in scaffold dimensions would be ideal to maintain good integration with surrounding tissue. Nevertheless, with the increasing MAA modification, the hydrogels became less elastic with poorer structural integrity due to the embrittlement effect. The samples disintegrated easily with poor handleability with the high percentage of MAA modification, probably due to the disruption of electrostatic and charged pair interactions of the collagen fibers (Morozova, S. and Muthukumar, M., The Journal of Chemical Physics, 2018, 149, 163333).

Furthermore, the degradation profile by the BCA assay demonstrated that high degradation rate was observed at both low and high MAA to collagen ratios (FIG. 3d), in which 1mL/g of MAA exhibiting an optimal stability and handleability over 21 days of study. Hence, an optimal 1 mL/g MAA to collagen sample was selected for further studies.

Example 3. Isolation and Characterisation of BFGel Samples

Isolation of Bullfrog Skin-derived Gelatins (BFGels)

Bullfrog skins were exposed to a temperature of 70 °C either under 0.5 M acetic acid (Type A gelatin) or neutral condition (Type B gelatin) over 48 hours upon cleaning with 0.5 M NaOH. Thereafter, centrifugation was carried out for pigmentation removal at 30,000 xg and 25 °C prior to the dialysis using 3.5 K MWCO SnakeSkin® dialysis tubing against 0.1 M acetic acid and distilled water for 48 h with a solution changed at 24 h time point. Lastly, the samples were lyophilized until further usage.

Attenuated Total Reflectance Fourier Transformation Infrared (ATR-FTIR) Spectroscopy Measurements of the BFGels

The isolated gelatin was first characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, in which the sample was placed directly onto the ATR sampling stage, followed by scanning in a range of 4000 - 650 cm ¹ with a resolution of 2 cm^-1 over 16 scans using a Frontier MIR/FTIR spectrometer (Perkin Elmer Inc., USA). Peaks were used to identify the characteristic amide groups of gelatin. Circular Dichroism Analysis

To evaluate the successful denaturation of the collagen into gelatin, circular dichroism (CD) was conducted to confirm the loss of the triple helix by following the protocol disclosed in Example 1.

Confirmation of Type A and Type B BFGel Samples via TNBS Assay

The successful isolation of Type A and Type B gelatin was validated through TNBS assay analysis, which was performed by following the protocol disclosed in Example 1. Generally, Type B gelatin has lower free amine groups, attributable to the oxidation of glutamate and aspartate to glutamic and aspartic acid during denaturation under strong alkaline conditions.

Results and Discussions

Prior to the fabrication of GelMA hydrogels, the isolated pristine BFGel samples (FIG. 4a) were first characterized. ATR-FTIR analysis revealed the typical characteristic amide peaks of gelatin (FIG. 4b). CD spectroscopy, which is a common technique used to assess the secondary structure as well as the folding properties of proteins, confirmed the full denaturation of collagen into gelatin products. CD indicated the disappearance of the triple helical structure, via the absence of the positive band at 222 nm for all gelatin samples (FIG. 4c). Further analysis by TNBS assay showed higher amount of free amine sides in Type A gelatin as compared to Type B (FIG. 4d), which agrees with earlier report by Liu et al. (International Journal of Biological Macromolecules, 2020, 162, 405) that preservation of glutamate and aspartate amino acid occurs during the acidic isolation process. Overall, the results showed that Type A and Type B BFGels were successfully extracted from the discarded bullfrog skin using both acidic and neutral conditions.

Example 4. Fabrication and Characterisation of Methacrylated Gelatin (GelMA) Precursors

Methacrylated gelatin (GelMA) precursors were prepared and characterized by following the protocol disclosed in Example 1.

Preparation of Methacrylated Gelatin (GelMA) Precursors

For the synthesis of methacrylated gelatin, either Type A or Type B GelMA, the respective BFGel was dissolved in DI water at 12% w/v, followed by pH stabilization using 1.5 M CB buffer overnight before the introduction of MAA at desirable MAA to gelatin ratio where the reaction was carried out at 50 °C over 4 days. Thereafter, the similar neutralization, dialysis using 3.5 K MWCO SnakeSkin® dialysis tubing, and lyophilization were carried out to produce GelMA.

Amino acid composition:

The amino acid compositions of bullfrog derived GelMA and a commercial porcine derived GelMA were determined by the hydrolysis of gelatin samples at a concentration of 20 mg/mL using 6M HCI in a controlled, inert environment at 110°C for 24 hours. After hydrolysis, the samples were filtered through a 0.45 pm syringe filter prior to five-fold dilution to achieve a final concentration of 4 mg/mL. 2 mL of this solution was then subjected to drying to evaporate the HCI solution using a rotary evaporator at 45°C, followed by reconstitution with sample buffer solutions at different pH for subsequent analysis. The amino acid composition was measured using a Biochrom Amino Acid Analyzer equipped with a Na-type cation exchange resin chromatography column. Post-separation on the column, the amino acids reacted with ninhydrin, and the resulting products were detected via spectrophotometry.

Results and Discussions

The isolated Type A and Type B BFGel were then subjected to the MAA modification at 1 mL/g ratio. TNBS assay confirmed the successful modification of GelMA with a reduction in the free amine groups (FIG. 5a). Between the two samples, higher amine functional groups were present in Type A GelMA as compared to Type B GelMA, although both were modified with same amount of MAA. This is due to the preservation of glutamate and aspartate amino acid during the acidic isolation of BFGel. On the other hand, for the isolation of Type B BFGel, oxidation of glutamine and asparagine into the respective glutamic acid and aspartic acid occurred to convert a portion of amine side chains into carboxyl groups (Baydin, T. et al., Food Hydrocolloids, 2022, 127, 107535).

Due to the preserved free amine functional sites of Type A GelMA with a higher degree of modification, Type A GelMA is strongly favored for creating stable hydrogels. Prior to hydrogel fabrication, the synthesized Type A GelMA was further analyzed alongside commercially available Type A GelMA from porcine origin to differentiate the obtained sample from the commercial products, for instance, the amino acid composition as shown in Table A. The amino acid compostions of the GelMA samples were found to be notably different. The BF GelMA was also found to exhibit a higher solubility limit of 60% w/v in water at room temperatue compared to the porcine GelMA, which was found to have a solubility limit of 15% w/v in water at room temperature. The high solulbility of BF GelMA in water could allow for use of a broader range of photocrosslinking formulations and provide better handleability, which can be a unique source of Type A bullfrog GelMA. Example 5. Fabrication and Characterisation of GelMA Hydrogels

Fabrication of GelMA Hydrogels

To fabricate the GelMA hydrogel, the procedures were similar to that of the ColMA (disclosed in Example 2), except that the lyophilized GelMA was first dissolved in deionised (DI) water at a 40% w/v concentration prior to mixing with either phenol red solution or SFM together with the LAP photoinitiator to form a complete precursor solution with a final GelMA concentration of 30% w/v for photocrosslinking process.

Characterisation of GelMA Hydrogels

The physicochemical properties of the GelMA hydrogels were evaluated by measuring the storage modulus, dimensional changes, and degradability according to the disclosed protocol in Example 2.

Results and Discussions

Type A and Type B GelMA hydrogels were successfully formed in the presence of LAP photoinitiator upon exposed to UV light (FIG. 6a).

Physicochemical analysis demonstrated that the GelMA exhibited significantly higher storage modulus as compared to the ColMA with similar modification ratio (FIG. 6b). This is likely due to the higher degrees of crosslinking observed in the gelatin samples, where the loss of its triple helical conformation enabled higher accessibility of the reactive functional groups.

In addition, minimal dimensional changes were observed in both GelMA hydrogels upon exposed to 1 x PBS due to the high crosslinking density in stabilising the overall structure that is ideal for tissue engineering applications (FIG. 6c).

The long-term stability of the fabricated hydrogels was evaluated over 21 days, in which Type A GelMA showed a slower degradation rate as compared to Type B due to the higher physical properties of the Type A GelMA hydrogel, thus indicating a better methacrylation efficiency was obtained in Type A GelMA (FIG. 6d).

Example 6. Cytocompatibility evaluation of the developed ColMA and GelMA Hydrogels

The potential of the developed hydrogels as cell encapsulant materials was evaluated using human keratinocytes (HaCaT) cells where the cells (5 x 1O⁶ cells/mL) were incorporated into the neutralised precursors prior to UV crosslinking. Thereafter, the viability of the HaCaT cells encapsulated within the hydrogels was examined under a fluorescence microscope via LIVE/DEAD staining.

Fabrication of the HaCaT-encapsulated Hydrogels

HaCaT cells (Division of Genetics of Skin Carcinogenesis, Germany Cancer Research Center) were cultured and expande’ in Dulbecco's Modified Eagle's Medium (DMEM; Sigma) supplemented with 10% FBS and 1 x Antibiotic-Antimycotic (Gibco®). Upon reaching confluency, HaCaT cells were trypsinized and counted before being added to the neutralized hydrogel precursors at 5 x 10⁶ cells/mL that containing either, ColMA or GelMA and LAP. Upon UV crosslinking, the viability of the HaCaT cells encapsulated within the hydrogels was examined under a fluorescence microscope via LIVE/DEAD staining.

Viability of the HaCaT-encapsulated Hydrogels

The hydrogels were incubated with staining solution consisting of 16 pL fluorescein diacetate (5 mg/ml), 16 pL propidium iodide (2 mg/ml) and 1 pL Hoechst (10 mg/mL) in 10 mL of SFM. The samples were incubated for 15 min prior to imaging using a Zeiss Axio Observer Z1 inverted fluorescence microscope (Carl Zeiss, Germany) fitted with a camera.

Results and Discussions

To evaluate the functionality of the developed hydrogels particularly as encapsulant materials, human keratinocytes (HaCaT) were encapsulated in the ColMA and GelMA hydrogels (1 mL/g of MAA to protein). Our preliminary results verified the hydrogels’ cytocompatibility. LIVE/DEAD staining of HaCaT cells encapsulated within ColMA and GelMA hydrogels on Day 0 demonstrated that majority of the cells were viable with good homogeneous distribution of cells within the hydrogels (FIG. 7). The survival of cells had verified that bullfrog collagen hydrogels were indeed possible as cell encapsulants.

Example 7. Modular Bioink for 3D Printing of Hybrid Hydrogel

Biomaterial selection for 3D bioprinting application is often associated with adjustability of the viscosity, stability/degradability, and rapid crosslinking time either as a scaffold or cell encapsulant material (Derakhshanfar, S. et al., Bioactive Materials, 2018, 3, 144). Here, we have designed a new bioink to print a tissue-mimicking hybrid material via Lumen X+ (the first digital light processing printer specifically designed for bioprinting with minimal dimensional changes), as shown in FIG. 8. Preparation of Photocrosslinkable Bioink for 3D Printing of Hybrid Hydrogel

This novel photocrosslinkable bioink comprises of pre-dissolved ColMA in 0.5M acetic acid, GelMA in DI water, and a LAP photoinitiator in high concentrations, which were then mixed and neutralized in various ColMA-to-GelMA ratios with a final 0.1 % w/v LAP (i.e., 0.75% w/v ColMA and 7.50% w/v GelMA, and another combination of bioink with 0.50% w/v ColMA and 15.00% w/v GelMA) aiming to produce hydrogels with varying properties upon photocrosslinking.

Results and Discussions

ColMA is capable of exhibiting high viscosity with preserved triple helical structures, which in tandem enable both cell-material interactions and homogeneous distribution of cells in the precursor solution during the bioprinting process. Despite the advantages of ColMA, the intrinsic self-assembly process of the collagen fibril often associated with high shrinkage of the obtained hydrogels, which could limit its application for tissue engineering (Sarrigiannidis, S O. et aL, Materials Today Bio, 2021 , 10, 100098). On the other hand, although GelMA has low viscosity, the fabricated hydrogel exhibits high modulus with minimal dimensional changes upon UV-crosslinking. Considering the advantages of ColMA and GelMA, hybrid hydrogels with different concentrations of ColMAand GelMA can be developed as an ideal bioink for 3D bioprinting application.

The newly developed bioink enables the printing of complex 3D constructs that are highly compatible with the human body. This could further enhance the scalability of 3D printed tissue manufacturing, with a wide array of applications in regenerative medicine.

Example 8. Stiffness Controlled Hybrid Hydrogels for Cell Behaviour Regulation

Fabrication of Hybrid Hydrogels

Hybrid hydrogels were fabricated by mixing ColMA (dissolved in 0.5M acetic acid) and GelMA (dissolved in DI water) in various proportions outlined in Table B. Subsequently, the mixture was neutralized, followed by the addition of LAP photoinitiator. The resulting mixture was then transferred into a mold and subjected to UV crosslinking to produce disc-shaped hydrogels, following the procedure outlined in Example 2 for assessing changes in modulus.

Cytocompatibitiliy and the Influence of Stiffness on Cell Morphology

HDFs were cultured and expanded in Dulbecco's Modified Eagle's Medium (DMEM; Sigma) supplemented with 10% FBS and 1 * Antibiotic-Antimycotic (Gibco®). Upon reaching confluency, HDFs were trypsinized and counted before being added to the respective hydrogel precursors at 5 * 10^A6 cells/mL. After crosslinking, the cells were cultured for 21 days before imaged according to Example 6 to visualize their morphology.

Results and Discussions

Initial findings indicated an increase in stiffness with higher proportions of GelMA (n = 1) attributed to GelMA's significantly higher concentration compared to ColMA, resulting in a greater net material content and providing sufficient physical support (FIG. 9).

The cytocompatibility and the influence of stiffness on cell morphology were investigated by encapsulating human dermal fibroblast cells (HDFs) as preliminary to observe their spreading capability, as the degree of cell spreading ultimately affects cellular behavior. A stiff pure GelMA (#5) and a softer hybrid hydrogel (#3) were used, and most of the cells remained viable as indicated by green fluorescence. Cells within the softer hydrogel (#3) spread well, while those within the stiffer part were confined to a rounded shape (FIG. 10). These observations are expected to influence cell behavior, particularly stem cells, which will differentiate into different lineages based on morphology.

Claims

1. A hydrogel precursor formulation comprising:

(b) a photoinitiator.

2. The hydrogel precursor formulation according to Claim 1, wherein both ColMA and GelMA are present.

3. The hydrogel precursor formulation according to Claim 2, wherein a weight to weight ratio of ColMA to GelMa is from 1 :7.5 to 1 :90, such as from 1 :10 to 1 :30, such as from 1 :10 to 1:20.

4. The hydrogel precursor formulation according to any one of the preceding claims, wherein the photoinitiator is selected from one or more of the group consisting of irgacure, and more particularly, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 2-hydroxy-1-[4-(2- hydroxyethoxy)phenyl]-2-methyl-1-propanone (lrgacure-2959), 2,2’-Azobis[2-Methyl-N-(2- hydroxyethyl) propionamide] (VA-086), and eosin Y (EY), optionally wherein the photoinitiator is LAP.

5. The hydrogel precursor formulation according to any one of the preceding claims, wherein the ColMA has a triple helical structure of collagen that is retained and is not denatured.

6. The hydrogel precursor formulation according to any one of the preceding claims, wherein the ColMA has a free amine group amount of from 15 to 50%, such as from 20 to 45%, such as from 25 to 35%.

7. The hydrogel precursor formulation according to any one of the preceding claims, wherein the ColMA, when formed into a hydrogel, has a storage modulus of from 1 to 4 kPa, such as from 2 to 3 kPa.

8. The hydrogel precursor formulation according to any one of the preceding claims, wherein the ColMA, when formed into a hydrogel, undergoes a shrinkage of from 15 to 30%, such as from 18 to 25%, such as about 20%.

9. The hydrogel precursor formulation according to any one of the preceding claims, wherein the GelMA has one or more of the following properties when present:

(ia) a free amine group amount of from 25 to 50%, such as from 25.5 to 45%;

(ib) a histidine count of one per 1 ,000 residues; and

(ic) a solubility limit of about 60% w/v in water at room temperature.

10. The hydrogel precursor formulation according to any one of the preceding claims, wherein the GelMA, when formed into a hydrogel, has one or more of the following properties:

(aa) a storage modulus of from 15 to 30 kPa, such as from 18 to 28 kPa;

(ab) a dimension change of from -5% to 5%, such as from -3% to 2%.

11 The hydrogel precursor formulation according to any one of the preceding claims, wherein a weight to weight ratio of the photoinitiator to:

(ba) ColMA is from 1 :2 to 1:10, such as from 1:5 to 1 :7.5; and/or

(be) GelMA is from 1:50 to 1 :300, such as from 1 :50 to 1 :200 or from 1 :75 to 1 :150.

13. The ColMa according to Claim 12, wherein the ColMA has one or more of the following properties:

14. A bioink comprising a hydrogel precursor formulation according to any one of Claims 1 to 11.

15. The bioink according to Claim 14, further comprising an aqueous solvent.

16. A kit of parts comprising:

(i) a methacrylated bullfrog collagen (ColMA) in an acidic aqueous medium;

(ii) a methacrylated gelatin (GelMA) in an aqueous medium; and (iii) a photoinitiator, optionally wherein the photoinitiator is selected from one or more of the group consisting of irgacure, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 2- hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (lrgacure-2959), 2,2’-Azobis[2- Methyl-N-(2-hydroxyethyl) propionamide] (VA-086), and eosin Y (EY), further optionally wherein the photoinitiator is LAP.

17. The kit of parts according to Claim 16, wherein one or more of the following apply:

(dh) the GelMA has a histidine count of one per 1 ,000 residues; and

18. A kit of parts comprising:

(ai) a methacrylated bullfrog collagen (ColMA) in an acidic aqueous medium; and

(aii) a photoinitiator, optionally wherein the photoinitiator is selected from one or more of the group consisting of irgacure, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 2- hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (lrgacure-2959), 2,2’-Azobis[2- Methyl-N-(2-hydroxyethyl) propionamide] (VA-086), and eosin Y (EY), further optionally wherein the photoinitiator is LAP.

19. The kit of parts according to Claim 18, wherein one or more of the following apply:

(ec) the ColMA, when formed into a hydrogel, has a storage modulus of from 1 to 4 kPa, such as from 2 to 3 kPa; (ed) the ColMA, when formed into a hydrogel, undergoes a shrinkage of from 15 to 30%, such as from 18 to 25%, such as about 20%.

20. A hydrogel formed by the crosslinking of a methacrylated bullfrog collagen (ColMA).

21. The hydrogel according to Claim 20, wherein the hydrogel has one or both of the following properties:

(fa) a storage modulus of from 1 to 4 kPa, such as from 2 to 3 kPa; and

22. Use of a hydrogel precursor formulation as described in any one of Claims 1 to 11 to encapsulate cells, optionally wherein the cells are living and remain in a viable state after encapsulation.

23. A method of encapsulating cells, the method comprising:

(ga) providing a mixture of a hydrogel precursor formulation according to any one of Claims 1 to 11 and cells; and

25. The GelMA according to Claim 24, wherein the GelMA has one or more of the following properties:

(ga) a free amine group amount of from 25 to 50%, such as from 25.5 to 45%;

(gb) a histidine count of one per 1 ,000 residues; and

(gc) a solubility limit of about 60% w/v in water at room temperature.

26. The GelMA according to Claim 24 or Claim 25, wherein the GelMA, when formed into a hydrogel, has one or more of the following properties: (aa) a storage modulus of from 15 to 30 kPa, such as from 18 to 28 kPa; and

(ab) a dimension change of from -5% to 5%, such as from -3% to 2%.

27. A kit of parts comprising:

(ib) a photoinitiator, optionally wherein the photoinitiator is selected from one or more of the group consisting of irgacure, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 2- hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (lrgacure-2959), 2,2’-Azobis[2- Methyl-N-(2-hydroxyethyl) propionamide] (VA-086), and eosin Y (EY), further optionally wherein the photoinitiator is LAP.

28. The kit of parts according to Claim 27, wherein one or more of the following apply:

(aad) the GelMA has a histidine count of one per 1 ,000 residues; and