Hydrogels

Abstract

A hydrogel comprising: water; an alginate; a glucono-delta-lactone (GDL); and microparticles comprising an inorganic calcium compound and recombinant gelatin. The hydrogels may be used for bone repair and/or regeneration.

Claims

1. A hydrogel comprising: water; an alginate; a glucono-delta-lactone (GDL); and microparticles comprising an inorganic calcium compound and recombinant gelatin.

2.-10. (canceled)

11. The hydrogel according claim 1 which becomes fluid when agitated and resolidifies when resting.

12. The hydrogel according to claim 1 comprising: a. 1 to 20 w/v % of the microparticles; b. 0.01 to 10 w/v % of inorganic calcium compound(s); c. 0.01 to 1% w/v % of the GDL; and d. 0.5 to 5 w/v % of the alginate.

13.-19. (canceled)

20. The hydrogel according to claim 1 wherein the gelatin is a recombinant gelatin cross-linked with formaldehyde, glutaraldehyde, hexamethylene diisocyanate, a carbodiimide, cyanamide and/or by dehydrothermal treatment.

21.-23. (canceled)

24. The hydrogel according to claim 1 wherein the microparticles have an average diameter of 1 μm to 1000 μm.

25.-27. (canceled)

28. The hydrogel according to claim 1 wherein the weight ratio of inorganic calcium compound to the recombinant gelatin is between 3:2 to 2:3.

29. (canceled)

30. The hydrogel according to claim 1 which further comprises one or more of a synthetic polymers, natural polymers, pharmaceutically active compounds, growth factors, proteins, crosslinkers or natural bone components.

31. The hydrogel according to claim 1 wherein the gelatin comprises glutamic acid and aspartic acid and the standard deviation (SD.sub.ED) distribution of the combined amounts of glutamic acid and aspartic acid in each row of 60 amino acids of said recombinant gelatin is at most 1.30.

32. The hydrogel according to claim 1 which is an injectable hydrogel.

33.-34. (canceled)

35. The hydrogel according claim 1 wherein the inorganic calcium compound is in the form of particles and the particles of the inorganic calcium compound are distributed randomly within recombinant gelatin particles.

36.-39. (canceled)

40. A process for preparing a hydrogel comprising the steps of precipitating an inorganic calcium compound in the presence of a recombinant gelatin to form microparticles comprising the inorganic calcium compound and the recombinant gelatin and mixing the so formed microparticles with a composition comprising water, alginate and GDL.

41. The process according to claim 48 which further comprises the step of hydrolysing at least a part of the GDL to form gluconic acid.

42. The medicament comprising a sealed bottle or ampoule and a hydrogel according to claim 1 wherein the hydrogel is present in the sealed bottle or ampoule.

43. The hydrogel according to claim 1 comprising: a. 1 to 20 w/v % of the microparticles; b. 0.01 to 10 w/v % of inorganic calcium compound(s); c. 0.01 to 1% w/v % of the GDL; and d. 0.5 to 5 w/v % of the alginate; wherein the gelatin comprises amino acids and at least 8% of each set of 60 amino acids of the recombinant gelatin are glutamic acid and/or aspartic acid.

44. The hydrogel according to claim 43 wherein the gelatin is a recombinant gelatin cross-linked with formaldehyde, glutaraldehyde, hexamethylene diisocyanate, a carbodiimide, cyanamide and/or by dehydrothermal treatment.

45. The hydrogel according to claim 43 wherein the weight ratio of inorganic calcium compound to the recombinant gelatin is between 3:2 to 2:3.

46. The hydrogel according to claim 43 wherein the gelatin comprises glutamic acid and aspartic acid and the standard deviation (SD.sub.ED) distribution of the combined amounts of glutamic acid and aspartic acid in each row of 60 amino acids of said recombinant gelatin is at most 1.30.

47. The hydrogel according to claim 43 wherein: (i) the gelatin is a recombinant gelatin cross-linked with formaldehyde, glutaraldehyde, hexamethylene diisocyanate, a carbodiimide, cyanamide and/or by dehydrothermal treatment; (ii) the weight ratio of inorganic calcium compound(s) to the recombinant gelatin is between 3:2 to 2:3; and (iii) the gelatin comprises glutamic acid and aspartic acid and the standard deviation (SD.sub.ED) distribution of the combined amounts of glutamic acid and aspartic acid in each row of 60 amino acids of said recombinant gelatin is at most 1.30.

48. The hydrogel according to claim 47 wherein the microparticles have an average diameter of 1 μm to 1000 μm.

Description

DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1 shows the morphology of a microparticle used in the hydrogels of the present invention.

[0047] FIGS. 2a to 2e show the rheological behavior of the hydrogels under different strains and the rupture of the hydrogels.

[0048] FIGS. 3a and 3b show the thixotropic behavior (viscous state under stress and self-healing after stress is removed) of the hydrogels composed of alginate, microspheres and GDL.

[0049] In FIGS. 2a to 2e and FIGS. 3a and 3b, G′ (square dots) is the storage (or elastic) modulus and Gii (triangular dots) is the loss (or viscous) modulus. More information is provided in the Examples section below.

[0050] The recombinant gelatin is preferably a non-fibrilar recombinant gelatin and preferably has a lower molecular weight than native gelatin. Furthermore, the recombinant gelatin preferably comprises glutamic and/or aspartic acid residues homogeneously distributed along the chain. Preferably the recombinant gelatin comprises a total amount of at least 8% glutamic and/or aspartic acids, e.g. per 60 amino acids in a row, with a standard deviation of at most 1.6. For the purpose of increasing the total calcium phosphate (or more specifically, hydroxyapatite) binding capacity, the absolute occurrence of glutamic and/or aspartic acid residues preferably is at least 9%, more preferably about 10%.

[0051] The recombinant gelatin preferably has an average molecular weight of less than 150 kDa, preferably of less than 100 kDa. Preferably the recombinant gelatin has an average molecular weight of at least 5 kDa, preferably at least 10 kDa and more preferably of at least 30 kDa. Preferred average molecular weight ranges for the recombinant gelatin include 50 kD to 100 kDa, 20 kDa to 75 kDa and 5 kDa to 40 kDa. Lower molecular weights may be preferred when higher mass concentrations of gelatins are required because of the lower viscosity.

[0052] The recombinant gelatin may be obtained commercially, e.g. from FUJIFILM under the tradename Cellnest™. The recombinant gelatin may also be prepared by known methods, for example as described in patent applications EP 0 926 543 and EP 1 014 176, the content of which is herein incorporated by reference. The methodology for preparing recombinant gelatins is also described in the publication ‘High yield secretion of recombinant gelatins by Pichia pastoris’, M.W.T. Werten et al., Yeast 15, 1087-1096 (1999). Suitable recombinant gelatins are also described in WO 2004/85473.

[0053] The amount of recombinant gelatin present in the hydrogel is preferably from 1 to 20 w/v %, more preferably 1 to 10 w/v %, especially 1.5 to 8 w/v %.

[0054] In one embodiment the recombinant gelatin comprises at least two lysine residues, said lysine residues being extreme lysine residues wherein a first extreme lysine residue is the lysine residue that is closest to the N-terminus of the gelatine and the second extreme lysine residue is the lysine residue that is closest to the C-terminus of the gelatine and said extreme lysine residues are separated by at least 25 percent of the total number of amino acids in the gelatin. Such recombinant gelatins may be obtained by, for example, the methods described in US 2009/0246282.

[0055] In another embodiment the gelatin is a the recombinant gelatin comprising at least two amino acid residues, said two amino acid residues being extreme amino acid residues, which independently are selected from an aspartic acid residue and a glutamic acid residue, wherein a first aspartic acid residue or glutamic acid residue is the aspartic acid residue or glutamic acid residue that is closest to the N-terminus of the polypeptide and the second extreme aspartic acid residue or glutamic acid residue is the aspartic acid residue or glutamic acid residue that is closest to the C-terminus of the polypeptide and said extreme aspartic acid residues and/or glutamic acid residues are separated by at least 25 percent of the total number of amino acids in the recombinant gelatin polypeptide.

[0056] In a preferred embodiment the recombinant gelatin has excellent cell attachment properties and preferably does not display any health-related risks.

[0057] The recombinant gelatin preferably has an isoelectric point of at least 5.

[0058] Preferably the recombinant gelatin is an RGD-enriched recombinant gelatin, e.g. a recombinant gelatin in which the percentage of RGD motifs related to the total number of amino acids is at least 0.4. If the RGD-enriched gelatin comprises 350 amino acids or more, each stretch of 350 amino acids preferably contains at least one RGD motif. Preferably the percentage of RGD motifs is at least 0.6, more preferably at least 0.8, more preferably at least 1.0, more preferably at least 1.2 and most preferably at least 1.5. A percentage RGD motifs of 0.4 corresponds with at least 1 RGD sequence per 250 amino acids. The number of RGD motifs is an integer, thus to meet the feature of 0.4%, a gelatin consisting of 251 amino acids should comprise at least 2 RGD sequences. Preferably the RGD-enriched recombinant gelatin comprises at least 2 RGD sequences per 250 amino acids, more preferably at least 3 RGD sequences per 250 amino acids, most preferably at least 4 RGD sequences per 250 amino acids.

[0059] The recombinant gelatin preferably comprises at least three RGD motifs. In a further embodiment an RGD-enriched gelatin comprises at least 4 RGD motifs, preferably at least 6, more preferably at least 8, even more preferably at least 12 up to and including 16 RGD motifs.

[0060] The recombinant gelatins used in this invention are preferably derived from collagenous sequences. Nucleic acid sequences encoding collagens have been generally described in the art. (See, e.g., Fuller and Boedtker (1981) Biochemistry 20: 996-1006; Sandell et al. (1984) J Biol Chem 259: 7826-34; Kohno et al. (1984) J Biol Chem 259: 13668-13673; French et al. (1985) Gene 39: 311-312; Metsaranta et al. (1991) J Biol Chem 266: 16862-16869; Metsaranta et al. (1991) Biochim Biophys Acta 1089: 241-243; Wood et al. (1987) Gene 61: 225-230; Glumoff et al. (1994) Biochim Biophys Acta 1217: 41-48; Shirai et al. (1998) Matrix Biology 17: 85-88; Tromp et al. (1988) Biochem J 253: 919-912; Kuivaniemi et al. (1988) Biochem J 252: 633640; and Ala-Kokko et al. (1989) Biochem J 260: 509-516).

[0061] Recombinant gelatins enriched in RGD motifs may also be prepared by, for example, the general methods described in US 2006/0241032.

[0062] An example of a suitable source of recombinant gelatin which may be used in the method of this invention is human COL1A1-1. A part of 250 amino acids comprising an RGD sequence is given in WO 04/85473. RGD sequences in the recombinant gelatin can adhere to specific receptors on cell surfaces called integrins.

[0063] RGD-enriched gelatins can be produced by recombinant methods described in, for example, EP-A-0926543, EP-A-1014176 or WO 01/34646, especially in the Examples of the first two mentioned patent publications. The preferred method for producing an RGD-enriched recombinant gelatin comprises starting with a natural nucleic acid sequence encoding a part of the collagen protein that includes an RGD amino acid sequence. By repeating this sequence an RGD-enriched recombinant gelatin may be obtained. Thus the recombinant gelatins can be produced by expression of nucleic acid sequence encoding such gelatins by a suitable micro-organism. The process can suitably be carried out with a fungal cell or a yeast cell. Suitably the host cell is a high expression host cells like Hansenula, Trichoderma, Aspergillus, Penicillium, Saccharomyces, Kluyveromyces, Neurospora or Pichia. Fungal and yeast cells are preferred to bacteria as they are less susceptible to improper expression of repetitive sequences. Most preferably the host will not have a high level of proteases that cleave the gelatin structure being expressed. In this respect Pichia or Hansenula offers an example of a very suitable expression system. Use of Pichia pastoris as an expression system is disclosed in EP 0 926 543 and EP 1 014 176. The microorganism may be free of active post-translational processing mechanism such as in particular hydroxylation of proline and also hydroxylation of lysine. Alternatively the host system may have an endogenic proline hydroxylation activity by which the gelatin is hydroxylated in a highly effective way.

[0064] In a further embodiment, the recombinant gelatin has less glycosylation than native gelatin, e.g. a glycosylation of less than 2 wt %, preferably less than 1 wt %, more preferably less than 0.5 wt %, especially less than 0.2 wt % and more especially less than 0.1 wt %. In a preferred embodiment the recombinant gelatin is free from glycosylation.

[0065] The degree or wt % of glycosylation refers to the total carbohydrate weight Thus the recombinant gelatins can be produced by expression of nucleic acid sequence encoding such gelatins by a suitable micro-organism. The process can suitably be carried out with a fungal cell or a yeast cell. Suitably the host cell is a high expression host cells like Hansenula, Trichoderma, Aspergillus, Penicillium, Saccharomyces, Kluyveromyces, Neurospora or Pichia. Fungal and yeast cells are preferred to bacteria as they are less susceptible to improper expression of repetitive sequences. Most preferably the host will not have a high level of proteases that cleave the gelatin structure being expressed. In this respect Pichia or Hansenula offers an example of a very suitable expression system. Use of Pichia pastoris as an expression system is disclosed in EP 0 926 543 and EP 1 014 176. The microorganism may be free of active post-translational processing mechanism such as in particular hydroxylation of proline and also hydroxylation of lysine. Alternatively the host system may have an endogenic proline hydroxylation activity by which the gelatin is hydroxylated in a highly effective way.

[0066] The degree or wt % of glycosylation preferably refers to the total carbohydrate weight per unit weight of the gelatin, as determined by, for example, MALDI-TOF-MS (Matrix Assisted Laser Desorption Ionization mass spectrometry) or by the titration method by Dubois. The term ‘glycosylation’ refers not only to monosaccharides, but also to polysaccharides, e.g. di- tri- and tetra-saccharides.

[0067] There are various methods for ensuring that glycosylation is low or absent. Glycosylation is a post-translational modification, whereby carbohydrates are covalently attached to certain amino acids of the gelatin. Thus both the amino acid sequence and the host cell (and enzymes, especially glycosyltransferases) in which the amino acid sequence is produced determine the degree of glycosylation. There are two types of glycosylation: N-glycosylation begins with linking of GlcNAc (N-actylglucosamine) to the amide group of asparagines (N or Asn) and O-glycosylation commonly links GalNAc (N-acetylgalactosamine) to the hydroxyl group of the amino acid serine (S or Ser) or threonine (T or Thr).

[0068] Glycosylation can, therefore, be controlled and especially reduced or prevented, by choosing an appropriate expression host, and/or by modifying or choosing sequences which lack consensus sites recognized by the host's glycosyltransferases. Chemical synthesis of gelatin can also be used to prepare gelatin which is free from glycosylation. Also recombinant gelatin which comprises glycosylation may be treated after production to remove all or most of the carbohydrates or non-glycosylated gelatin may be separated from glycosylated gelatin using known methods.

[0069] Surprisingly recombinant gelatins described above give rise to efficient nucleation and growth of low-crystalline inorganic calcium compound particles.

[0070] Preferably the microparticles are obtained by precipitating the inorganic calcium compound in the presence of the recombinant gelatin. For example, one may dissolve the recombinant gelatin in an aqueous solution (e.g. at a concentration typically between 1% and 30%), acidify the solution (e.g. using for example carbonic or phosphoric acid) and mixing the resultant solution with calcium hydroxide (e.g. by adding the acidified recombinant gelatin solution to a solution of calcium hydroxide). It is also possible to first mix the recombinant gelatin with a calcium source (e.g. calcium hydroxide solution) and subsequently add the carbonic or phosphoric acid. It is further possible to add the inorganic calcium compound as a fine powder to an aqueous solution of the recombinant gelatin.

[0071] After precipitating or mixing the inorganic calcium compound in the presence of the recombinant gelatin (typically allowing a crystallization process to occur in the presence of the recombinant gelatin) a composite slurry is usually obtained.

[0072] To increase the biomimetic character of the hydrogels of the present invention the Inorganic calcium compound may further comprise additives such SO.sub.3.sup.2−, Na.sup.+, Mg.sup.2+, Sr.sup.2+, Si4.sup.+, Zn.sup.2+, SiO.sub.4.sup.4− and/or HPO.sub.4.sup.2− ions. In one embodiment the hydrogels of the present invention comprise one or more of such additives in a total amount of 0.01% to 25 wt %. Especially preferred are additive concentrations that mimic the amounts of such additives in natural, human bone. Preferably the gelatin is a cross-linked recombinant gelatin because this can increase the storage stability of the hydrogel. Crosslinking is preferably achieved using cross-linkable groups, e.g. carboxy or amino groups, present in the recombinant gelatin. Techniques for crosslinking gelatin are already described in literature. Mostly crosslinking occurs through the carboxylic acid or amine groups of the gelatin.

[0073] The crosslinking agent which may be used is not particularly limited. For example one may use a chemical crosslinking agent, e.g. formaldehyde, glutaraldehyde, hexamethylene diisocyanate, carbodiimides and/or cyanamide.

[0074] Preferably the crosslinking does not impair the biocompatibility of the microparticles in the hydrogel and does not generate a strong immune response. In that respect, the use of dehydrothermal treatment as a crosslinking method is preferred. Also the use of hexamethylene diisocyanate as a crosslinking agent is preferred.

[0075] In a preferred embodiment the hydrogel comprises: [0076] a. 1 to 20 w/v % of the microparticles; [0077] b. 0.01 to 10 w/v % of the inorganic calcium compound(s); [0078] c. 0.01 to 1% w/v % of the GDL (or combined amount of GDL and gluconic acid); and [0079] d. 0.5 to 5 w/v % of the alginate.

[0080] According to a second aspect of the present invention there is provided a process for preparing a hydrogel comprising the steps of precipitating an inorganic calcium compound in the presence of a recombinant gelatin to form microparticles comprising the inorganic calcium compound and the recombinant gelatin and mixing the so formed microparticles with a composition comprising water, alginate and GDL.

[0081] The process preferably further comprises hydrolysing at least a part of the GDL to form gluconic acid, preferably by exposing the GDL to biological conditions of a human or animal body.

[0082] According to a third aspect of the present invention there is provided a process for preparing a hydrogel comprising injecting into a human or animal body a composition comprising:

[0083] water;

[0084] an alginate;

[0085] a glucono-delta-lactone (GDL); and

[0086] microparticles comprising an inorganic calcium compound and recombinant gelatin.

[0087] In the second and third aspects of the present invention the preferred inorganic calcium compound, recombinant gelatin, GDL and alginate are as described herein in relation to the first aspect of the present invention.

[0088] According to a fourth aspect of the present invention there is provided a medicament comprising a sealed bottle or ampoule and a hydrogel according to the first aspect of the present invention, wherein the hydrogel is present in the sealed bottle or ampoule.

[0089] The invention will now be illustrated with the following non-limiting Examples.

EXAMPLES

Materials:

[0090]

TABLE-US-00001 Material Source Hexamethylene diisocyanide Sigma-Aldrich (St. Louis, MO, USA) (HMDIC) (>98.0% purity) Corn Oil Sterile Sodium Chloride Calcium Carbonate (CaCO.sub.3) fine powder (average particle size < 1 μm) GDL Ethanol Millipore (Billerica, MA, USA) Acetone Hydrochloric Acid Pronova SLM 20 (sterile Novamatrix (Sandvika, Norway) alginate where over 50% of the monomer units are mannuronate) Pronova SLG 20 (sterile alginate where over 60% of the monomer units are guluronate) rhBMP-2% A 2 wt % solution of mature re- combinant human bone morphogenetic protein-2 (rhBMP-2 peptide) containing amino acids of BMP-2 283 to 396 plus an N-terminal Met- Ala. Expressed in E. coli, isolated from inclusion bodies, renatured and purified as described by Kirsch T, Nickel J, Sebald W (Isolation of recombinant BMP receptor IA ectodomain and its 2:1 complex with BMP-2. FEBS letters. 2000; 468: 215-9) Gel Type A native pigskin-derived gelatin (i.e. not recombinant and therefore outside the scope of the present claims).

Preparation of Recombinant Gelatins

[0091] Recombinant gelatins (SEQ ID NO: 1 and 2) were prepared based on a nucleic acid sequence that encodes for a part of the gelatin amino acid sequence of human COLIAI-I and modifying this nucleic acid sequence using the methods disclosed in EP-A-0926543, EP-A-1014176 and WO01/34646. The gelatins did not contain hydroxyproline and comprised the amino acid sequences identified herein as in SEQ ID NO: 1 or 2. Except for the last incomplete row, the total amount of GLU+ASP per row of 60 amino acids is shown on the right side of each row.

TABLE-US-00002 number of (GLU+ASP) residues per 60 amino acids in a row: SEQ ID NO: 1 GAPGAPGLQGAPGLQGMPGERGADGLPGPKGERGDAGPKG 6 ADGAPGAPGLQGMPGERGAA GLPGPKGERGDAGPKGAAGAPGKDGVRGLAGPIGPPGERG 7 AAGLPGPKGERGDAGPKGAD GAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAG 5 LPGPKGERGDAGPKGADGAP GKDGVRGLAGPPGAPGLQGAPGLQGMPGERGAAGLPGPKG 5 ERGDAGPKGADGAPGAPGLQ GMPGERGAAGLPGPKGERGDAGPKGAAGAPGKDGVRGLAG 6 PIGPPGERGAAGLPGPKGER GDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQG 6 MPGERGAAGLPGPKGERGDA GPKGADGAPGKDGVRGLAGPPGAPGLQGAPGLQGMPGERG 6 AAGLPGPKGERGDAGPKGAD GAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPG 6 KDGVRGLAGPIGPPGERGAA GLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAG 6 APGAPGLQGMPGERGADGLP GPKGERGDAGPKGADGAPGKDGVRGLAGPPG SEQ ID NO: 2 GAPGAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKG 4 AAGAPGAPGLQGMPGERGAA GLPGPKGERGDAGPKGAAGAPGKDGVRGLAGPIGPPGERG 6 AAGLPGPKGERGDAGPKGAA GAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGARGADG 5 LPGPKGERGDAGPKGADGAP GKAGVRGLAGPPGAPGLQGAPGLQGMPGARGAAGLPGPKG 1 ARGDAGPKGAAGAPGAPGLQ GMPGERGAAGLPGPKGERGDAGPKGAAGAPGKDGVRGLAG 6 PIGPPGERGAAGLPGPKGER GDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQG 6 MPGERGAAGLPGPKGERGDA GPKGADGAPGKDGVRGLAGPPGAPGLQGAPGLQGMPGERG 5 AAGLPGPKGARGDAGPKGAD GAPGAPGLQGMPGARGAAGLPGPKGERGDAGPKGADGAPG 5 KDGVRGLAGPIGPPGERGAA GLPGPKGERGDAGPKGAAGAPGKAGVRGLAGPIGPPGPAG 3 APGAPGLQGMPGERGAAGLP GPKGERGDAGPKGADGAPGKDGVRGLAGPPG

[0092] The distribution of GLU+ASP in the gelatin is represented by the standard deviation of the amounts per row (see Table 1 below).

[0093] SEQ ID NO: 1 and 2 were used to prepare the various hydrogels described in the Examples below.

TABLE-US-00003 TABLE 1 Amount/distribution of (GLU + ASP) in SEQ ID NO: 1 and 2. Average Number of Standard deviation (GLU + ASP) (GLU + ASP) amount % Amount of residues per 60 per 60 amino acids in gelatin (GLU + ASP) amino acids in row row SEQ ID NO: 1 9.8 5.9 0.60 SEQ ID NO: 2 9.8 5.9 1.69

Step 1) Preparation of Microparticles by Precipitation of CaCO.SUB.3 .in the Presence of Gelatins.

[0094] A 20% aqueous (20 g of gelatins SEQ ID NO: 1 or NO: 2 or Type A pigskin derived gelatin) solution was prepared and mixed with CaCO.sub.3 fine powder (with a size of <1 μm) in a 1:1 (w/w) ratio of gelatin to CaCO.sub.3. This suspension was emulsified in corn oil at 50° C. while stirring the emulsion at 800 rpm for 20 min. After cooling down the emulsion, the emulsified microparticles were washed three times with acetone. After overnight drying at 60° C., microparticles were sieved to 50-72 μm size using sieves (Retsch GmbH, Haan, Germany). Particles were crosslinked for each gelatin using hexamethylene diisocyanide (HMDIC) or dehydrothermal crosslinking (DHT), as indicated in Table 2 below.

Crosslinking with HMDIC: 1 g of spheres and 1 mL of HMDIC (>98.0% pure, Sigma)) were mixed in 100 ml ethanol for 1 day. Excess cross-linker was removed by washing several times with ethanol.
Crosslinking by DHT: 1 g of spheres were crosslinked at 160° C. in vacuum (˜5.10.sup.−3 mbar) oven for 4 days.

[0095] In Comparative Example 10, the inorganic calcium compound (CaCO.sub.3) was removed from the microparticles by treating the microparticles with excess hydrochloric acid (1M, Merck) until carbon dioxide formation stopped. The calcium-free microparticles were then washed 3 times with deionised water and subsequently dried overnight drying at 60° C.

[0096] All crosslinked particles were gamma sterilized afterwards by Synergy Health (Etten Leur, The Netherlands) prior to use in in vitro and in vivo experiments.

Step 2) Loading of the Microparticles with Excipients

[0097] Microparticles prepared in step 1 (68 mg) were incubated with 170 μL rhBMP-2 at a concentration of 122.5 μg/mL at 4° C. overnight.

[0098] For Examples 13, 14 and CEx7, 136 mg were incubated with 170 μL rhBMP-2 at a concentration of 122.5 μg/mL.

Step 3) Preparation of the Hydrogels

[0099] Two alginate solutions were prepared based on alginate SLM20 or SLG20 by adding 0.9% sterile sodium chloride to create 2% w/v alginate (SLM or SLG) solutions.

[0100] The microparticles from step 2 were added to 1014 μL of 2% w/v of alginate SLM20 or 1014 μL of 2% w/v alginate SLG20. Immediately 106 μL of 0.06M fresh glucono delta lactone (GDL) solution was added and mixed.

[0101] Also Comparative Examples CEx8 and CEx9 were prepared in which the microparticles and BMP-2 were omitted. When required additional 0.9% sterile sodium chloride was used to ensure that the Comparative Examples had the same alginate concentration as the actual Examples.

[0102] Comparative Example CEx8 was prepared mixing 1014 μL of 2% w/v SLM with 276 μL of 0.25% calcium chloride.

[0103] Comparative Example CEx9 was prepared by mixing 34 mg CaCO.sub.3, 170 μL 0.9% sodium chloride, 1014 μL of 2% w/v alginate SLM20 and 106 μL of 0.06M fresh glucono delta lactone (GDL).

[0104] Comparative Example CEx10 was prepared by adding the microparticles from step 1 without CaCO.sub.3 to 1014 μL of 2% w/v of alginate SLM20 and mixing 34 mg CaCO.sub.3, and 106 μL of 0.06M fresh glucono delta lactone (GDL).

[0105] The resultant hydrogels were as described in Table 2 below.

[0106] In Table 2: [0107] SLM means Pronova SLM 20 (an alginate) and % is the w/v %, i.e. the weight of alginate relative to the total volume of the composition [0108] SLG means Pronova SLG 20 (an alginate) and % is the w/v %, i.e. the weight of alginate relative to the total volume of the composition [0109] MS % means the w/v % (i.e. the weight of microparticles relative to the total volume of the composition) of the relevant microparticle derived from the Gelatin (Gel) and CaCO.sub.3 indicated in the next two columns of Table 2; [0110] Gel ID/% shows the gelatin used (SEQ1=SEQ ID NO: 1, SEQ2=SEQ ID NO: 2; and Gel=Type A pigskin (Comparative).

TABLE-US-00004 TABLE 2 Hydrogels and Comparative Hydrogels Showing the Percentage (w/v) of each Component Alginate Example ID/% MS % Gel ID/(%) CaCO.sub.3% GDL % rhBMP-2 % Crosslinking MS 1 SLM/1.5 5.2 SEQ1/2.6% 2.6% 0.089% 0.0016% HMDIC 2 SLM/1.5 5.2 SEQ2/2.6% 2.6% 0.089% 0.0016% HMDIC CEx1 SLM/1.5 5.2  Gel/2.6% 2.6% 0.089% 0.0016% HMDIC 3 SLG/1.5 5.2 SEQ1/2.6% 2.6% 0.089% 0.0016% HMDIC 4 SLG/1.5 5.2 SEQ2/2.6% 2.6% 0.089% 0.0016% HMDIC CEx2 SLG/1.5 5.2  Gel/2.6% 2.6% 0.089% 0.0016% HMDIC 5 SLM/1.5 5.2 SEQ1/2.6% 2.6% — 0.0016% HMDIC 6 SLM/1.5 5.2 SEQ2/2.6% 2.6% — 0.0016% HMDIC CEx3 SLM/1.5 5.2  Gel/2.6% 2.6% — 0.0016% HMDIC 7 SLG/1.5 5.2 SEQ1/2.6% 2.6% — 0.0016% HMDIC 8 SLG/1.5 5.2 SEQ2/2.6% 2.6% — 0.0016% HMDIC CEx4 SLG/1.5 5.2  Gel/2.6% 2.6% — 0.0016% HMDIC 9 SLM/1.5 5.2 SEQ1/2.6% 2.6% — 0.0016% DHT 10  SLM/1.5 5.2 SEQ2/2.6% 2.6% — 0.0016% DHT CEx5 SLM/1.5 5.2  Gel/2.6% 2.6% — 0.0016% DHT 11  SLM/1.5 5.2 SEQ1/2.6% 2.6% 0.089% 0.0016% DHT 12  SLM/1.5 5.2 SEQ2/2.6% 2.6% 0.089% 0.0016% DHT CEx6 SLM/1.5 5.2  Gel/2.6% 2.6% 0.089% 0.0016% DHT 13  SLG/1.5 10.4 SEQ1/2.6% 5.2% 0.089% 0.0016% HMDIC 14  SLG/1.5 10.4 SEQ2/2.6% 5.2% 0.089% 0.0016% HMDIC CEx7 SLG/1.5 10.4  Gel/2.6% 5.2% 0.089% 0.0016% HMDIC CEx8 SLM/1.5 — — — — — — CEx9 SLM/1.5 — — 2.6% 0.089% — —  CEx10 SLM/1.5 2.6 SEQ1/2.6%  2.6%* 0.089% 0.0016% HMDIC *in CEx10 the CaCO3 was removed_from the microparticles by treating the microparticles with excess hydrochloric acid, as described above.

Evaluation of the Hydrogels and Comparative Hydrogels Described in Table 2

[0111] The mechanical properties of prepared hydrogels were measured by a Rheometer (Anton Paar MCR301, Graz, Austria). A 20 mm diameter parallel plate measuring system was used. After sample addition to the plate, silicon oil was applied to the edges to prevent evaporation. Storage (or elastic) modulus (G′) and loss (or viscous) modulus (G″) were measured between 0%-400% strain at 37° C. to assess the viscoelastic region. To study the thixotropic behaviour, a different setting was used which included two-step repeating cycle. At the first step of the cycle, storage and loss moduli were measured at 1% strain, at 1 Hz, at 37° C. At the second step, 500% strain, 1 Hz frequency, 37° C. temperature was applied. The cycle was repeated several times to characterize thixotropic behavior. Normal force was set to 0.1 N. Strain-dependent oscillatory rheology of gelatin microparticle alginate hydrogels, as shown in Table 3, showed an extremely broad linear viscoelastic region in addition to network rupture at high strains at 150% for Example 7, Example 8 and Comparative Example 4 hydrogels. The mechanical properties were increased with addition of GDL that rupture occurs for Examples 1 to 4 and Comparative Examples CEx1 and CEx2 at a strain of >170% (Table 3). This showed the importance of GDL in the composition. The highest strain for network rupture was observed for formulations Examples 11 and 12 and

[0112] Comparative Example CEx6 which contained DHT crosslinked microparticles shows a network rupture at very high strain (>350), indicated that composites containing DHT crosslinked microparticles have higher mechanical properties.

[0113] The Comparative Example CEx8 hydrogel (without microparticles) broke at about 12% strain. In Comparative Example CEx9 the hydrogel did not contain microparticles but contained 1 μm CaCO.sub.3 crystals and broke at about 40% strain. Also Comparative Example CEx10 in which the microparticles were free from inorganic calcium compounds (the calcium compounds were removed as described above) and to which 34 mg CaCO.sub.3 had been added had a low rupture strain of only 35%. The results shown in Table 3 show the importance of the hydrogels of the invention having the claimed features.

TABLE-US-00005 TABLE 3 Strain % at which Example the structure breaks Comparative Examples CEx8, CEx9 12-40 and CEx10 Examples 9 to 12 and Comparative >350 Examples CEx5 and CEx6 Examples 1 to 4 and Comparative >170 Examples CEx1 and CEx2 Examples 7 and 8 and Comparative 150 Example CEx4

[0114] The hydrogels of Examples 1 to 8 and Comparative Examples CEx1 to CEx4 and Examples 13 and 14 and Comparative Example CEx7 possessed self-thinning behaviour under stress and self-recovery of the hydrogel after the stress had been removed. This behaviour proves that the gel will be reformed in situ directly after injection in vivo. The formed hydrogels showed good mechanical properties as it could be seen from their storage (or elastic) moduli. When stress was removed, the storage moduli were between 1-2 kDa in both alginate gel formulations which were comparable to that of endothelial tissue and stromal tissue.

Cell Attachment on In Situ Gelling Hydrogels

[0115] C2C12 cells (muscle fibroblast mouse cells CRL-1772 from ATCC) were cultured at 37° C. and 5% CO.sub.2 in DMEM (Dulbecco's modified eagle's medium from Invitrogen) media supplemented with 10% fetal bovine serum (FBS) (Sigma) and 1% penicillin-streptomycin (Sigma).HG compositions were prepared as described above. From these formulations, 200 μL of was added to each well of 24-well-plates.

[0116] C2C12 cells were seeded on hydrogels as 4750 cells/well. After 5 days of cell seeding, cells were stained with Live/Dead (Invitrogen) mixture for approximately 45 min. After staining, cells were visualized under fluorescent light by Olympus BX60 light microscope. The results by visual inspection of the samples (see table 4) show that cells preferably attached to microparticles inside the hydrogel formulation rather than only gel. Further it is shown that RGD containing recombinant gelatin having enhance cell attachment in vitro.

TABLE-US-00006 TABLE 4 cell attachment by visual inspection of C2C12 cells Visual inspection 1 ++ 2 ++ CEx1 + 3 ++ 4 ++ CEx2 + 5 ++ 6 ++ CEx3 + 7 ++ 8 ++ CEx4 + 9 ++ 10  ++ CEx5 + 11  ++ 12  ++ CEx6 + 13  +++ 14  +++ CEx7 + CEx8 − CEx9 −  CEx10 ++ +++is best and −is worst