EXTRACELLULAR MATRIX MATERIAL

Abstract

An extracellular matrix material is described. The material has a cross-linked scaffold comprising fibrin or fibrinogen, and a bulking agent. Deposited on the scaffold is a calcium phosphate mineral phase. Also described are methods for forming such materials.

Claims

1. An extracellular matrix material, comprising: a cross-linked scaffold comprising fibrin or fibrinogen, and a bulking agent, and a ceramic deposited on the scaffold.

2. An extracellular matrix according to claim 1, wherein the ceramic is, or comprises, a calcium phosphate mineral phase.

3. An extracellular matrix material according to claim 1 or claim 2, wherein the bulking agent is an alginate, such as sodium alginate or a derivatised alginate, such as sodium propyiglycoalginate.

4. An extracellular matrix material according to claim 1 or claim 2, wherein the bulking agent is a glycosaminoglycan (GAG; e.g. chondroitin 6-sulfate, chondroitin 4-sulfate, heparin, heparin sulphate, keratan sulfate, dermatan sulfate, chitin, chitosan, dextran sulphate or hyaluronan).

5. An extracellular matrix material according to claim 1 or claim 2, wherein the bulking agent is selected from hydroxyethylstarch, ethyl cellulose, Xanthan gum and agarose.

6. A process for preparing an extracellular matrix material, comprising: depositing a ceramic on a cross-linked scaffold comprising fibrin or fibrinogen, and a bulking agent.

7. A process according to claim 6, wherein the ceramic is a calcium phosphate mineral phase.

8. A process according to claim 6 or claim 7, wherein the calcium phosphate mineral phase is deposited on the scaffold by biomimetic deposition.

9. A process according to any of claims 6 to 8, wherein the calcium phosphate mineral phase is deposited on to the scaffold by immersing the scaffold in a fluid comprising Ca.sup.2+ and HPO.sub.4.sup.2, optionally further comprising: i) Na.sup.+, K.sup.+, Mg.sup.2+, Ca.sup.2+, Cl.sup., HCO.sub.3.sup., HPO.sub.4.sup.2 and SO.sub.4.sup.2; ii) Na.sup.+, K.sup.+, Mg.sup.2+, Ca.sup.2+, Cl.sup., HCO.sub.3.sup. and HPO.sub.4.sup.2; or iii) Na.sup.+, K.sup.+, Ca.sup.+.

10. A process as defined in any of claims T to 9, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 1 to 30 mM Ca.sup.2+, preferably 1 to 20 mM Ca.sup.2.

11. A process as defined in any of claims 7 to 10, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 0.5 to 10 mM HPO.sub.4.sup.2.

12. A process as defined in any of claims 7 to 11, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 100 to 800 mM Na.sup.+.

13. A process as defined in any of claims 7 to 12, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 1 to 50 mM K.sup.+.

14. A process as defined in any of claims 7 to 13, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 0.5 to 20 mM Mg.sup.2+.

15. A process as defined in any of claims 7 to 14, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 100 to 800 mM Cl.sup..

16. A process as defined in any of claims 7 to 15, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 1 to 100 mM HCO.sub.3.sup..

17. A process as defined in any of claims 7 to 16, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 1 to 10 mM SO.sub.4.sup.2.

18. A process as defined in any of claims 7 to 17, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 1 to 5 mM Ca.sup.2+, preferably 2 to 3 mM Ca.sup.2+, most preferably about 2.5 mM Ca.sup.2+.

19. A process as defined in any of claims 7 to 18, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 0.5 to 3 mM HPO.sub.4.sup.2 preferably 0.5 to 2 mM HPO.sub.4.sup.2, most preferably, about 1 mM of HPO.sub.4.sup.2.

20. A process as defined in any of claims 7 to 19, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 100 to 200 mM Na.sup.+, preferably 120 to 160 mM Na.sup.+, even more preferably 140 to 150 mM of Na.sup.+, most preferably about 142 mM Na.sup.+.

21. A process as defined in any of claims 7 to 20, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 1 to 10 mM K.sup.+, preferably 3 to 7 mM K.sup.+, most preferably, about 5 mM K.sup.+.

22. A process as defined in any of claims 7 to 21, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 0.5 to 3 mM Mg.sup.2+, preferably 1 to 2 mM Mg.sup.2+, most preferably about 1.5 mM Mg.sup.2+.

23. A process as defined in any of claims 7 to 22, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 100 to 200 mM Cl.sup., preferably 120 to 160 mM Cl.sup., more preferably 140 to 150 mM of Cl.sup., most preferably about 148.8 mM Cl.sup..

24. A process as defined in any of claims 7 to 23, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 1 to 10 mM HCO.sub.3.sup., preferably 2 to 8 mM HCO.sub.3.sup., more preferably 4 to 5 mM HCO.sub.3.sup., most preferably about 4.2 mM HCO.sub.3.sup..

25. A process according to any of claims 11 to 24, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 1 to 5 mM Ca.sup.2+, 0.5 to 3 mM HPO.sub.4.sup.2, 100 to 200 mM Na.sup.+, 1 to 10 mM K.sup.+, 0.5 to 3 mM Mg.sup.2+, 100 to 200 mM Cl.sup. and 1 to 10 mM HCO.sub.3.sup..

26. A process according to any of claims 11 to 25, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 2 to 3 mM Ca.sup.2+, 0,5 to 2 mM HPO.sub.4.sup.2-, 120 to 160 mM Na.sup.+, 3 to 7 mM K.sup.+, 1 to 2 mM Mg.sup.2+, 120 to 160 mM Cl.sup. and 2 to 8 mM HCO.sub.3.sup..

27. A process as defined in any of claims 7 to 17, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 10 to 30 mM Ca.sup.2+.

28. A process as defined in any of claim 7 to 17 or 27, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 5 to 10 mM HPO.sub.4.sup.2.

29. A process as defined in any of claims 7 to 17 or 27 to 28, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 500 to 800 mM Na.sup.+.

30. A process as defined in any of claims 7 to 17 or 27 to 29, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 10 to 40 mM K.sup.+.

31. A process as defined in any of claims 7 to 17 or 27 to 30, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 5 to 15 mM Mg.sup.2+.

32. A process as defined in any of claims 7 to 17 or 27 to 31, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 500 to 800 mM Cl.sup..

33. A process as defined in any of claims 7 to 17 or 27 to 32, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 10 to 100 mM HCO.sub.3.sup..

34. A process as defined in any of claims 7 to 17 or 27 to 33, wherein the calcium phosphate mineral phase is deposited on to the scaffold by contacting the scaffold with, or immersing the scaffold in, a fluid comprising 1 to 10 mM SO.sub.4.sup.2.

35. A process according to any of claims 9 to 34, wherein the scaffold is contacted with, or immersed in, the fluid for at least 12 hours, at least 24 hours, at least 2 days, at least 5 days, at least 6 days, or at least 9 days.

36. A process according to any of claims 9 to 35, wherein the scaffold is contacted with, or immersed in the fluid for up to 20 days, preferably up to 10 days, most preferably up to 9 days.

37. A process according to any of claims 7 to 36, wherein the scaffold has been obtained, has been formed, or is obtainable by a process comprising: (a) mixing an aqueous solution of fibrinogen with a coagulating agent and a bulking agent; (b) incubating the mixture obtained in step (a) with a cross-linking agent; and (c) washing the cross-linked composition obtained in step (b) to remove the cross-linking agent.

38. A process according to any of claims 7 to 36, comprising forming the scaffold by a process comprising the following steps: (a) mixing an aqueous solution of fibrinogen with a coagulating agent and a bulking agent; (b) incubating the mixture obtained in step (a) with a cross-linking agent; and (c) washing the cross-linked composition obtained in step (b) to remove the cross-linking agent.

39. A process according to claim 37 or claim 38, wherein fibrinogen is present at a purity level of greater than one of 75%, 80%, 85%, 90%, 95%, 97% or 99%.

40. A process according to any of claims 37 to 39, wherein fibrinogen is present as truncated forms of fibrinogen, such as fibrin A, fibrin B, fibrin C, fibrin D, fibrin X and fibrin Y.

41. A process according to claim 40, wherein the truncated form of fibrinogen is fibrin E.

42. A process as defined in any of claims 37 to 41 wherein fibrinogen is present as an aqueous solution buffered to a pH of between 4 and 10 with phosphate buffered saline (PBS) or HEPES buffered saline.

43. A process as defined in any of claims 37 to 42, wherein the coagulating agent comprises an enzymatic or non-enzymatic coagulating agent.

44. A process as defined in claim 43, wherein the coagulating agent is thrombin, such as human thrombin

45. A process as defined in any of claims 37 to 44, wherein step (a) comprises mixing with a foaming agent.

46. A process according to claim 45 wherein the foaming agent comprises a non-ionic detergent, thermosensitive gelling surfactant (e.g. pluronic 127) , diphosphatydylglycerol type phospholipid or a mixture of an immiscible phase with the aqueous fibrinogen solution phase, preferably wherein the foaming agent comprises a pluronic surfactant e.g. pluronic F68 and/or pluronic F127, more preferably pluronic F68.

47. A process as defined in claim 45 or claim 46, wherein the foaming agent is consists of or comprises one or more surfactant agent(s) from the class of sugar surfactants.

48. A process according to claim 47, wherein the foaming agent consists of or comprises one or more surfactant agents(s) from the class of sugar-acyl surfactants.

49. A process according to claim 48, wherein the foaming agent is from the class of sugar acyl surfactants having an acyl chain length from C.sub.8 to C.sub.12.

50. A process according to any of claims 47 to 49, wherein the foaming agent comprises or consists of at least two, preferably three sugar surfactants.

51. A process according to any of claims 48 to 50, wherein the foaming agent sugar-acyl surfactants are selected from the class of pyranoside (particularly glucopyranoside), maltoside, and acyl-sucrose surfactants.

52. A process according to any of claims 48 to 51, wherein the sugar-acyl surfactants are selected from the group consisting of OGP, ODM, DGP and DdGP, TOP, HGP, DMP, decyl sucrose (nDS), dodecylsucrose (nDdS), preferably OGM, DMP and DdGP.

53. A process according to any of claims 37 to 52, wherein the cross-linking agent used in step (b) is selected from: carbodiimide coupling agents such as N-(3-dimethylaminopropyl)-N-ethylcarbodiiniide (EDC); N-hydroxysuccinimide (NHS), azide coupling agents; diisocyanate cross-linking agents such as hexamethylene diisocyanate; epoxide cross-linking agents such as epi-chlorhydrin, glycidylethers and glycidylamines; and aldehyde cross-linking agents such as formaldehyde, glutar aldehyde and glyoxal.

54. A process as defined in claim 53, wherein the cross linking agent comprises an aldehyde cross-linking agent such as formaldehyde, glutaraldehyde and glyoxal.

55. A process as defined in claim 54, wherein the aldehyde cross-linking agent is glutaraldehyde.

56. A process according to any of claims 37 to 55, which additionally comprises addition of a reducing agent or a toxicity reducing agent, such as sodium borohydride or lysine.

57. A process according to any of claims 37 to 56, wherein the mixing step (a) is achieved by foaming, such as mixing with aeration.

58. A process according to any of claims 37 to 57, wherein the mixture obtained in step (a) is cast, frozen and optionally lyophilised prior to the incubation step (b).

59. A process according to any of claims 37 to 58, which additionally comprises addition of a divalent or multivalent metal ion such as calcium, such as calcium chloride.

60. A process according to any of claims 37 to 59, wherein the mixture for mixing step (a) further comprises a sugar as a protein stabilizer, wherein the sugar is a small polyol or carbohydrate, such as glycerol, sorbitol, sucrose or trehalose.

61. A process according to claim 60, wherein the sugar is trehalose, preferably in an amount of 10-11% wt. with respect to fibrinogen, and preferably in an amount of 4-7.5% wt. in the mixture of step (a).

62. A process according to any of claims 37 to 61 for preparing an extracellular matrix composition having a predetermined shape, wherein either (i) the mixture of step (a) is cast in a mould of a predetermined shape, frozen and optionally lyophilised prior to the incubation step (c), or; (ii) the product of step (d) is produced in a mould of a predetermined shape, and the product is then frozen and optionally lyophilised.

63. A process according to any of claims 37 to 62, wherein the bulking agent is an alginate, such as sodium alginate or a derivatised alginate, such as sodium propylglycoalginate.

64. A process according to any of claims 37 to 63, wherein the bulking agent is a glycosaminoglycan (GAG; e.g. chondroitin 6-sulfate, chondroitin 4-sulfate, heparin, heparin sulphate, keratan sulfate, dermatan sulfate, chitin, chitosan, dextran sulphate or hyaluronan).

65. A process according to any of claims 37 to 64, wherein the bulking agent is selected from hydroxyethylstarch, ethyl cellulose, Xanthan gum and agarose.

66. An extracellular matrix material obtained or obtainable by a process as defined in any of claims 6 to 65.

67. An extracellular matrix material according to any of claim 1 to 5 or 66, wherein the calcium phosphate mineral phase is at least 10%, preferably at least 20%, more preferably at least 50%, by weight, of the extracellular matrix material,

68. Use of an extracellular matrix composition as defined in claim 1 to 5 or 66 for use in the manufacture of a medicament for in vitro, ex vivo or in vivo bone regeneration or for treatment of a bone defect.

69. A method of bone regeneration which comprises application of an extracellular matrix material as defined in claim 1 to 5 or 66 to a bone defect such as a wound or fracture.

70. An extracellular matrix material according to any of claim 1 to 5 or 66 for use in bone regeneration in vitro, ex vivo or in vivo or for treatment of a bone defect.

71. An extracellular matrix material according to any of claims any of claim 1 to 5 or 66, wherein the calcium phosphate mineral phase comprises calcium orthophosphate compounds, preferably hydroxyapatite.

72. A process according to any of claims 7 to 65, wherein the calcium phosphate mineral phase comprises calcium orthophosphate compounds, preferably hydroxyapatite.

73. An extracellular matrix material according to any of claim 1 to 5 or 66, wherein the calcium phosphate mineral phase comprises octacalcium phosphate.

74. An extracellular matrix material according to any of claim 1 to 5, 66 or 73, wherein the calcium phosphate mineral phase comprises an amorphous and/or crystalline calcium phosphate mineral phase.

75. A process according to any of claims 7 to 65, wherein the calcium phosphate mineral phase comprises octacalcium phosphate.

76. A process according to any of claims 7 to 65, wherein the calcium phosphate mineral phase comprises an amorphous and/or crystalline calcium phosphate mineral phase.

Description

EXAMPLE 1

Materials and Methods

[0129] Preparation of Smart Matrix

[0130] The manufacturing process of Smart Matrix takes place in 3 distinct stages:

[0131] 1) vigorous mixing (4000 rpm) of pre-warmed (37 C.) reagents to form a white foam that is cast into a mould and incubated at 37 C. for 1 h to allow clotting

[0132] 2) chemical crosslinking with 0.2% vol/vol glutaraldehyde (Sigma-Aldrich, UK) in 80% absolute ethanol/20% MES buffer for 4 h at room temperature, followed by 1 wash with diH.sub.2O (10 min), 1 wash with 0.1% wt/vol NaBH.sub.4 (Sigma-Aldrich, UK) in diH.sub.2O (overnight), 5 washes with 0.1% wt/vol NaBH.sub.4 in diH.sub.2O (10 min), and 5 washes with diH.sub.2O (10 min) to eliminate any possible residue of glutaraldehyde and its reducing agent NaBH.sub.4

[0133] 3) lyophilisation of the scaffolds at 40 C. for 36 h (Virtis Genesis Freeze Dryer, Biopharma, UK),

[0134] Reagents mixed in the first stage are: [0135] 3 ml of 2% wt/vol alginate (Novamatrix, Norway) in MES buffer pH=7.4

[0136] 25 l of 1M CaCl.sub.2 (Sigma-Aldrich, UK) in diH.sub.2O

[0137] 6 ml of 2% wt/vol dialysed human fibrinogen (RiaSTAP, CSL Behring Ltd., UK) (Sharma et al. 2015) or bovine fibrinogen (Sigma-Aldrich, UK) in MES/NaCl buffer pH=7.4

[0138] 750 l of surfactant mix [20% wt/vol Pluronic F-68 (Sigma-Aldrich, UK) in diH.sub.2O, 20% wt/vol decyl--d-maltopyranoside (Sigma-Aldrich, UK) in diH.sub.2O, 20% wt/vol dodecyl--d-glucopyranoside (Sigma-Aldrich, UK) in diH.sub.2O, and 20% wt/vol octyl--d-maltopyranoside (Sigma-Aldrich, UK) in diH.sub.2O] (WO2013164635 A1). [0139] 1.2 ml of 10I.U./ml human thrombin (TISSEEL, Baxter International Inc., US) or dialysed bovine thrombin (Sigma-Aldrich, UK) in HEPES/NaCl buffer pH 7.4 [0140] 1.5 ml of 66% wt/vol trehalose (Fisher Scientific, UK) in diH.sub.2O [0141] Preparation of Calcium Phosphate-Smart Matrix (CaP-SM) by Immersion in Simulated Body Fluid (SBF):

[0142] SBF solution was prepared according to the protocol described by Kokubo et al. 1990. SBF solution contained a defined concentration of relevant ions (Table 1):

TABLE-US-00001 TABLE 1 Concentration of relevant ions in SBF solution. Ion Na.sup.+ K.sup.+ Mg.sup.2+ Ca.sup.2+ Cl.sup. HCO.sub.3.sup. HPO.sub.4.sup.2 mM 142 5 1.5 2.5 148.8 4.2 1

[0143] To prepare the SBF solution, the following quantities and reagents were added to 0.4 L of dH.sub.2O: 0.152 g of MgCl.sub.2-6H2O; 0.176 g of NaHCO.sub.3; 0.114 g of K.sub.2HPO.sub.4.3H2O; 0.139 g of CaCl.sub.2; 0.112 g of KCl and 4.027 g of NaCl. The solution was vigorously stirred for 10 min. The pH was adjusted to 7.25 with (CH.sub.2OH).sub.3CNH.sub.2 50 mM/HCl 45 mM buffer. Finally, the volume was topped up to 0.5 L with dH.sub.2O and the solution was vigorously stirred for 10 min.

[0144] 1 cm1 cm Smart Matrix scaffolds were immersed in SBF for up to 9 days at 37 C. SBF solution was changed every 2-3 days. After the immersion time, scaffolds were removed from the SBF solution, washed with dH.sub.2O, frozen at 80 C. and lyophilised. [0145] Scanning Electron Microscopy (SEM) and Energy Dispersive X-Ray Spectroscopy (EDX)

[0146] SEM and EDX analysis were conducted at the Nanovision Centre at Queen Mary's University of London.

[0147] Lyophilised CaP-SM samples after 0, 2, 4, 6 and 9 days of immersion in SBF were gold sputtered coated before observation under SEM and EDX analysis.

[0148] SEM microphotographs were taken at 150, 500, 2,000, 10,000, 20,000 and 40,000 magnifications and obtained at 5 kV. Morphology of scaffolds, qualitative pore size, morphology and size of CaP crystal deposits were studied from the SEM images. Elemental analysis of the CaP-SM scaffolds and Ca/P ratio were studied from the EDX spectra that were obtained at 10 kV. [0149] Von Kossa Staining of Cross-Sections:

[0150] CaP-SM scaffolds were embedded in paraffin and cross-sections cut to a thickness of 4 m using a microtome.

[0151] The principle of the Von Kossa staining is a precipitation reaction in which silver ions react with phosphate under acidic conditions. Then, photochemical degradation of silver phosphate to silver occurs under light illumination. A 1.5% silver nitrate solution was prepared by adding 1.5 g of AgNO.sub.3 to 100 ml of dH.sub.2O. Similarly, a 2.5% sodium thiosulphate solution was prepared by adding 2.5 g of Na.sub.2S.sub.2O.sub.3 to 100 ml of dH.sub.2O.

[0152] Slides were covered with 1.5% silver nitrate solution and expose to bright light for 1 h (under a lamp), after which they were washed with dH.sub.2O. Then slides were covered with 2.5% sodium thiosulphate for 5 min and dipped in running water before immersion in Eosin counter stain for 5 min. Slides were dipped in 70% IMS, then 90% IMS and immersed in 100% IMS for 1 min. Finally, they were immersed in Xylene for 2 min, dipped twice in Xylene, left to dry and coversliped for observation under light microscopy. CaP deposits were stained black/dark brown while Smart Matrix was stained pink/red. [0153] In Vitro Bio-Degradability (Trypsin Digestion)

[0154] 1 cm1 cm CaP-SM scaffolds (n=3 per time of immersion in SBF) were placed in a 12 well plate. To each well containing a scaffold disc 2.5 ml of a 0.25% trypsin in versene solution was added and the plate incubated at 37 C. with 5% CO.sub.2. At 0, 2, 4, 6, 24, 48, and 168 h (7 days) a 100 l aliquot from each well was transferred to an eppendorf tube and stored at 80 C. Each well was replenished with 100 l of fresh 0.25% trypsin in versene solution.

[0155] A total protein colorimetric assay was performed on the 100 l aliquots. A standard curve of bovine serum albumin in PBS from 0.1 to 1.5 mg/ml was prepared. Aliquots were allowed to thaw before 50 L from the aliquots and standards were mixed with 750 l of BradfordUltra solution in a cuvette. Following the manufacturer's instructions, absorbance at 595 nm was measured against air in a M550 double beam UV/visible spectrophotometer (Spectronic Camspec Ltd., UK). Absorbance at 595 nm for the 0 time point was subtracted from the other samples. A standard curve was plotted and used to calculate the percentage of matrix protein in solution considering that, if completely degraded, a 11 cm piece of the scaffolds would have 4 mg/ml of protein in solution. [0156] Cell Viability by AlamarBlue Assay:

[0157] Primary normal human dermal fibroblasts from three different donors were used. Cells were isolated and cultured as previously described (Sharma et al. 2015).

[0158] CaP-SM in SBF for 0, 2, 6 and 9 days were used for this study as SEM shows that after 2 days of immersion in SBF CaP deposits are up to 0.2 pm in size while at days 6 and 9 they are 0.05-0.8 m-larger aggregates. Besides, more CaP is observed after 9 days in SBF than after 6 days. Therefore, the effect of CaP deposits size and CaP concentration on cell viability were observed in this study.

[0159] 1 cm1 cm CaP-SM scaffolds were transferred to 24 well plates along with plastic coverslips (monolayer control) and washed once with IMS followed by 4 with PBS. 3 scaffolds per cell population were used, therefore n=9.

[0160] 0.510.sup.6 cells were seeded per scaffold at passage 5 and after 3 h incubation at 37 C. with 5% CO.sub.2 to allow the cells to attach to the scaffolds, 2 mL of medium were added per well.

[0161] At days 1, 2, 4, 7 and 10 of culture an alamarBlue assay was performed: 1 ml of alamarBlue working solution (diluted 10 from stock solution with phenol free DMEM) were added per well and samples incubated at 37 C. with 5% CO.sub.2 for 4 h, after which each well content was transferred to a cuvette and absorbance measured at 570 nm in a uv/vis spectrophotometer.

[0162] Results were statistically analysed by one-way ANOVA using Sigma Stat 3.5 software. A p<0.05 was considered a significant result. [0163] Cell Migration through the CaP-SM

[0164] Seeded CaP-SM scaffolds after 10 days of culture were fixed in 4% paraformaldehyde and processed for paraffin histology. Cross-sections were cut to a thickness of 4 pm using a microtome.

[0165] Sections were stained with a classic Haematoxylin & Eosin staining, which stains cells purple/black while the background appears bright pink.

[0166] Stained sections were dried and coverslipped for observation under light microscopy.

EXAMPLE 2

Results

[0167] SEM and EDX Analysis

[0168] SEM images in FIG. 1 show that Smart Matrix is a uniform and homogeneous material composed of fibres. It possesses micro (30-350 m) as well es nano-porosity (0.06-0.6 m). Pores appear to he interconnected.

[0169] SEM images displayed FIG. 2 show mineral deposition over time: after 2 days of immersion in SBF globular mineral deposits of up to 0.2 m in size are observed: after 4 days of immersion in SBF globular mineral crystals are observed on the surface of the fibrin/alginate matrix, which grow with increasing immersion time; after 6 days of immersion in SBF globular mineral crystals (0.05-0.8 m-larger aggregates) are observed on the entire surface for the biomaterial.

[0170] Although an increase in crystal size is seen with increasing immersion time in SBF solution, fibrin fibres are still visible after 9 days of immersion in SBF, as it can be seen from FIG. 2 bottom right. This suggests that the beneficial pro-angiogenic and bioactive properties of fibrin would not be masked by the presence of the crystals.

[0171] FIG. 3 images show low magnification photos of CaP deposition over time, suggesting that the original micro-porosity found in Smart Matrix is reduced upon immersion in SBF, as the reduction is noticeable after only 2 days of immersion in SBF (FIG. 3, top left):

[0172] EDX analysis (FIG. 4) of CaP-SM after 6 days of immersion in SBF shows that Ca and P are the main elements of the globular crystals, also at day 9. Before day 6 no Ca and P are detected in the EDAX spectra. Calculated Ca/P was 1.70.6 (averagestandard deviation; Ca/P for hydroxyapatite is 1.67). Mg is also present: SBF contains Mg.sup.2+ and Mg.sup.2+ is among the reported substituting ions present in bone mineral (Wopenka and Pasteris 2005; LeGeros 2008). [0173] Von Kossa Staining of Cross-Sections

[0174] FIG. 5 shows eosin staining of Smart Matrix scaffold used (left) and a Von Kossa stained scaffold, counterstained with eosin (right). As can be seen, Smart Matrix appears darker after the Von Kossa staining. The homogeneous open pore structure of the scaffold can also be seen as already observed in the SEM photos:

[0175] FIG. 6 shows the Von Kossa staining results of CaP-SM scaffolds after immersion in simulated body fluid (SBF) for up to 9 days. Samples were counterstained with eosin. CaP deposits are stained black/dark brown while Smart Matrix stains pink/red.

[0176] As observed from the photographs, the scaffold fibres appear darkened with immersion in SBF. Black/brown staining seems to increase with longer immersion periods in SBF. It is also interesting to note that the CaP deposits are found throughout the scaffold structure, which appears homogeneously stained. These results suggest that even deposition of CaP deposits throughout Smart Matrix can be achieved with a biomimetic procedure of immersion of scaffolds in SBF. [0177] In Vitro Bio-Degradability (Trypsin Digestion)

[0178] FIG. 7 shows that the in vitro degradation rate of the scaffolds decreases as the immersion time in SBF increases. The degradation rate is especially decreased in the first 6 h when compared to Smart Matrix. These results suggest that CaP-SM would take longer to degrade in vivo than Smart Matrix, which has been observed in histological sections up to 5-6 weeks after implantation in a full thickness skin wound in a porcine model. It has been suggested that new bone formation in vivo takes up to 4 months (Hadjidakis and Androulakis 2006), so a slower degradation of CaP-SM is expected to be beneficial for bone repair. [0179] Cell Viability by AlamarBlue Assay

[0180] Results displayed in FIG. 8 show that cells are able to grow and proliferate on the CaP-SM scaffolds, suggesting that the CaP deposited by the biomimetic method is not toxic to the cells.

[0181] There is a significant difference (p<0.05) between the monolayer control and cells on scaffolds until day 4, at day 7 only for CaP-SM 2 and 6 and then no significant difference at day 10. This may be due to an inefficient cell seeding on the 3D scaffolds compared to the flat control where the seeding efficiency is of 100%. As the culture period progresses cells in the 3D scaffolds proliferate more than on the flat control. Finally, there is no statistical differences between Smart Matrix and the CaP-SM scaffolds, suggesting that the beneficial cellular properties of Smart Matrix are still present in CaP-SM scaffolds. [0182] Cell Migration through the CaP-SM

[0183] Photographs in FIG. 9 show that primary normal human dermal fibroblasts were able to migrate through the CaP-SM scaffolds and after 10 days of culture populated the scaffolds throughout.

EXAMPLE 3

Materials and Methods for Forming Alternative Scaffolds

[0184] Alternative scaffolds were formulated by immersing Smart Matrix@ in SBF solutions as described below.

[0185] Table 2 describes the concentration of relevant ions present in the SBF solutions:

TABLE-US-00002 TABLE 2 Concentration of relevant ions in SBF solution. Solution Ion Na.sup.+ K.sup.+ Mg.sup.2+ Ca.sup.2+ Cl.sup. HCO.sub.3.sup. SO.sub.4.sup.2 HPO.sub.4.sup.2 SBF-1 mM 766.3 35.9 10.9 18.8 740.9 71.5 3.6 7.1 SBF-2 mM 686.1 31.2 9.3 15.6 728.6 26.1 6.2 SBF-3 mM 660 12.4 15.6 691.3 6.2

[0186] To prepare the SBF-1 solution, the following quantities and reagents were added to 0.7 L of dH.sub.2O: 1.465 g of CaCl.sub.2; 1.555 g of MgCl.sub.2.6H.sub.2O; 2.520 g of NaHCO.sub.3 and 1.150 g of K.sub.2HPO.sub.4.3H.sub.2O. Following this, the pH of the solution was adjusted to 6.0 using 1M HCl, after which, 0.360 g of Na.sub.2SO.sub.4, 1.125 g of KCl 27.015 g of NaCl and 2.130 g of Na.sub.2CO.sub.3 were added to the solution. Finally, the pH of the solution was adjusted to 6.5 using 1M HCl.

[0187] To prepare the SBF-2 solution, the following quantities and reagents were added to 0.4 L of dH.sub.2O: 0.695 g of CaCl.sub.2; 0.760 g of MgCl.sub.2.6H.sub.2O; 0.880 g of NaHCO.sub.3 and 0.570 g of K.sub.2HPO.sub.4.3H.sub.2O. Following this, the pH of the solution was adjusted to 6.0 using 1M HCl, after which, 0.560 g of KCl and 20.135 g of NaCl were added to the solution. Finally, the pH of the solution was adjusted to 6.5 using 1M NaOH.

[0188] To prepare the SBF-3 solution, the following quantities and reagents were added to 0.4 L of dH.sub.2O: 20.135g of NaCl; 0.695 g of CaCl.sub.2 and 0.570 g of K.sub.2HPO.sub.4.3H.sub.2O. The pH of this solution was adjusted to 6.0 with 1M HCl.

[0189] All solutions were filtered with 0.22pm PES membrane before use.

[0190] 1cm1 cm Smart Matrix scaffolds were immersed in either SBF-1 or SBF-2 solution for 24 hours with constant stirring at 160 rpm on an orbital shaker at 37 C. After the immersion time, scaffolds were removed from the SBF solutions, washed with dH.sub.2O in an ultrasonic water cleaner for 60 seconds, frozen at 80 C. and lyophilised.

[0191] Lyophilised scaffolds can be further immersed in SBF-3 for 48 hours with constant stirring at 80 rpm on an orbital shaker at 37 C. After the immersion time, scaffolds are removed from the SBF-3 solution, washed with dH.sub.2O in an ultrasonic water cleaner for 60 seconds, frozen at 80 C. and lyophilised.

[0192] Key: [0193] SM1: Smart Matrix scaffolds immersed in dH.sub.2O for 24 h (Control for CaP-SM 1). [0194] SM2: Lyophilised SM1 scaffolds immersed in dH.sub.2O for 48 h (Control for CaP-SM 2). [0195] CaP-SM1: Smart Matrix scaffolds immersed in SBF-1 for 24 h. CaP mineral deposits present a globular morphology. [0196] CaP-SM2: Lyophilised SmartCaP1 scaffolds further immersed in SBF-3 for 48 h. CaP mineral deposits present a plate-like morphology.

EXAMPLE 4

Methods & Results for Testing the Alternative Scaffolds

[0197] a) Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDX)

[0198] Results in FIG. 10 show CaP mineral deposits with a globular morphology distributed evenly throughout the fibrin matrix in CaP-SM1. A plate-like morphology is observed in CaP-SM2. SM1 and SM2 show the typical fibres seen in Smart Matrix.

[0199] EDX elemental analysis (FIG. 11) showed that the main elements in the mineral deposits of CaP-SM1 and CaP-SM2 were Ca and P. Calculated Ca/P was 1.290.28 and 1.020.37 (averagestandard deviation) for CaP-SM1 and CaP-SM2, respectively. Mg was also present in CaP-SM1 spectra. [0200] b) Fourier Transform Infrared Spectroscopy (FTIR) and X-Ray Diffraction (XRD)

[0201] Analysis of functional groups in the CaP deposits was carried out by FTIR. Spectra were obtained by placing the scaffolds in contact with Attenuated Total Reflectance accessory (Golden Gate ATR, Specac, UK). Spectrum software v 5.0.1 (Perkin-Elmer, UK) identified the peak intensities of each chemical group (the wavenumber was fixed between 500-4000 cm.sup.1 with a resolution of 4 cm.sup.1).

[0202] Phase composition and crystallinity of the CaP deposits were studied by XRD using a RIGAKU D/max 2500 Diffractometer operated at 40 kV and 80 mA with graphite-filtered Cu Ka radiation. Data was collected from 28=5 to 80 with a step size of 0.03.

[0203] FTIR spectra displayed in FIG. 12 show that differences exist between the controls (SM1 and SM2) and the CaP-SM scaffolds. PO.sub.4 burst peaks can be seen for the CaP-SM samples at approximately 1050 cm.sup.3 followed by a small shoulder peak at approximately 850 cm.sup.1 which could be due to a HPO.sub.4 group or a CO.sub.3 group or both. A peak at approximately 600 cm.sup.1, specially visible for CaP-SM2, could be due to a PO.sub.4 group. The small peak seen at approximately 1450 cm.sup.1 for CaP-SM1 could be due to a CO.sub.3 group. Moreover, spectra show that PO.sub.4 peaks grow in intensity in CaP-SM2 compared to CaP-SM1 suggesting deposition of a higher amount of a phosphate mineral phase in CaP-SM2 than in CaP-SM1 scaffolds. FTIR results suggest the presence of PO.sub.4, and possibly both HPO.sub.4 and CO.sub.3 groups, especially in CaP-SM1, in the CaP deposits.

[0204] XRD spectra of CaP-SM1 did not show peaks as the CaP deposits in these scaffolds are very amorphous and XRD does not detect very amorphous mineral phases. For CaP-SM2 XRD spectra revealed peaks that could correspond to hydroxyapatite (HA) and/or octacalcium phosphate (OCP). However, these peaks were broad indicating the CaP mineral phase/s present in CaP-SM2 were not very crystalline. Taking into account that the Ca/P ratio calculated by EDX is approximately 1, CaP-SM2 could be composed of a combination of CaP phases, for example OCP and amorphous calcium phosphate (ACP): Ca/P=1.33 and Ca/P=0.67-1.5 for OCP and ACP, respectively (Habraken et al. 2016). [0205] c) Von Kossa Staining of Cross-Sections

[0206] FIG. 13 shows Von Kassa stained scaffolds counterstained with eosin. Although it may not readily be visible from FIG. 13, CaP mineral deposits are stained black/dark brown while the fibrin fibres stain pink/red. As observed from FIG. 13, CaP mineral deposits are found throughout the scaffold structure suggesting an even deposition of CaP throughout the scaffolds. No CaP mineral deposits were observed on the SM1 and SM2 control samples. [0207] d) Rheology

[0208] Rheology is a branch of engineering that studies the viscoelastic properties (both solid and fluid) of materials as well as biological tissues (Holt etal. 2008; Saitoh etal. 2010). A Kinexus Rheometer (Malvern Instruments, UK) in an oscillatory mode was used. From each scaffold 2 cm2 cm samples were prepared and placed between two 20 mm diameter parallel plates (gap between plates=0.3 mm). The sample was hydrated and an integrated temperature controller was used to maintain the temperature of the sample stage at 20 C. A combined measurement including an amplitude sweep and a frequency sweep was carried out on each sample. The amplitude sweep was performed by applying controlled stresses that were linearly increased from 0.05 to 5%. Strains corresponding to the stresses were recorded. The oscillatory frequency was maintained at 1 Hz. The maximum strain within the linear viscoelastic region (LVER) was chosen from the amplitude sweep. The shear or storage modulus G was calculated for the different samples. G is related to elasticity and is an indication of how the material stores energy which can be re-used in the form of elastic deformation. Thus, G relates to the solid characteristics of the material.

[0209] Calculated G for the different scaffolds (as meanstandard error mean) was 11.242.54 kPa for SM1, 11.042.33 kPa for SM2, 75.2255.40 kPa for CaP-SM1 and 561.33109.79 kPa for CaP-SM2. Therefore, adding a CaP mineral phase to the fibrin-based matrix strengthens the material. A plate-like morphology makes the scaffold stronger than a globular morphology. [0210] e) In Vitro Bio-Degradability (Trypsin Digestion)

[0211] Scaffolds (Smart Matrix, demineralised bone matrix or DBM, CaP-SM 1 and CaP-SM 2) were cut into 5 mm5 mm square pieces prior to treatment with 1% trypsin at pH 7.2 at 37 C. for up to 48 h hours. Scaffolds were immersed in PBS alone as controls. Samples were imaged macroscopically using canon camera and microscopically using a stereomicroscope at 0 h, 18 h, 24 h and 48 h.

[0212] Images in FIG. 14 show that after 48 h both Smart Matrix and DBM are completely degraded while pieces of CaP-SM 1 and CaP-SM 2 are still visible. CaP-SM 1 degraded to a greater extent than CaP-SM 2 after 48 h in 1% Trypsin. These results suggest that CaP-SM scaffolds would take longer to degrade in vivo than Smart Matrix or DBM. [0213] f) Cell Viability and Proliferation by Live/Dead and AlarmarBlue Assays

[0214] Scaffolds (Smart Matrix used as control, CaP-SM 1 and CaP-SM 2) were cut into 5 mm5 mm square pieces and sterilised with 70% IMS, washed three times with PBS and placed in flat bottomed-24 well ultra-low attachment plates. The scaffolds were seeded with 110 MC3T3-E1 subclone mouse pre-osteoblasts (osteoprogenitor cells) in 20 l medium. After seeding, the plates were incubated for 3 h at 37 C. with 5% CO, to allow cells to attach to the scaffolds. After the attachment incubation time, 1 ml of MEM (Minimum Essential Medium Eagle Alpha Modification with 1% antibiotics and 10% fetal calf serum) with (+OM) or without (OM) osteogenic supplements (50 g/ml ascorbic acid, 10 mM -glycerophosphate and 100 nM dexamethasone) was added per well and cultured over a 28 day period at 37 C. with 5% CO.sub.2.

[0215] Seeded scaffolds were assessed for cell incorporation and viability using Live/Dead cell staining according to the manufacturer's guidelines (Sigma), wherein live cells fluoresce green and dead cells fluoresce red. Briefly, scaffolds were washed in PBS prior to staining with the live/dead staining solution and then the staining procedure was performed in the dark for 30 min at 37 C. and 5% CO.sub.2. Live and dead cells were visualized by fluorescence imaging and confocal microscopy.

[0216] Seeded scaffolds were transferred to fresh 24 well plates and 1 ml of alamarBlue working solution (diluted 10 from stock solution with phenol free Dubelcco's Modified Eagles Medium supplemented with 10% FCS and 1% antibiotics) was added per well and samples incubated at 37 C. with 5% CO.sub.2 for 3 h, after which each well content was transferred to a cuvette and absorbance measured at 570 nm in a uv/vis spectrophotometer.

[0217] Results showed that cells remained viable over the culture period and were able to proliferate. The Live/Dead assay showed green fluorescent cells (viable) on all scaffolds both under standard and osteogenic conditions (FIG. 15). Images also show that cells grow in clusters in CaP-SM scaffolds compared to Smart Matrix. Cells seemed to initially proliferate faster in Smart Matrix control and CaP-SM1 scaffolds until reaching a plateau at day 14 while in CaP-SM 2 cells did not reach a plateau in growth over the culture period (FIG. 16). These results suggest that CaP-SM 1 and CaP-SM 2 keep the beneficial cellular properties of Smart Matrix. [0218] g) Cell Migration through the CaP-SM

[0219] Seeded CaP-SM scaffolds after 28 days of culture were fixed in 4% paraformaldehyde, processed for paraffin histology and stained with standard Haematoxylin & Eosin (H&E) as in Example 1.

[0220] Images in FIG. 17 show that MC3T3-E.1 cells were able to migrate through the CaP-SM scaffolds and after 28 days of culture populated the scaffolds throughout. Smart Matrix has been shown to promote influx and migration of cells both in vitro and in vivo (Garcia-Gareta et al. 2013; Sharma et al. 2016) and these results show that CaP-SM scaffolds retain this property.

[0221] Additionally, distribution of MC3T3-E1 cells within Smart Matrix@ , CaP-SM 1 and CaP-SM2 scaffolds was examined using DAPI staining of the nuclei after 28 days of culture under standard (OM) and osteogenic (+OM) conditions. Histological slides were deparaffinised, rehydrated and washed in distilled water before Fluoroshield with DAPI mounting media (F6057 Sigma) was applied as per manufacturer's instructions. Briefly, the vial was brought to room temperature and 1-2 drops of the mounting media was applied directly on top of the sample and left to set for approximately 5 minutes at room temperature. The sections were then cover slipped carefully avoiding air bubbles and the edges were sealed using a nail varnish. The sections were then imaged using confocal scanning laser microscope using 405 nm wavelength laser for DAPI (Excitation 372, Emission 456). The sections were tile scanned for the entire XY plane of the scaffold and then merged using 5% overlap automated function of the confocal microscope.

[0222] Images in FIG. 18 show that cells populate the scaffolds throughout and are evenly distributed through the depth of the scaffolds. [0223] h) Cell Integration

[0224] After 28 days in culture either under standard or osteogenic conditions, seeded scaffolds were fixed with 2.5% glutaraldehyde. Prior to and post-fixing in 1% osmium tetraoxide (Sigma-Aldrich) for 1 h, the samples were washed in 0.1 M sodium cacodylate buffer. This was followed by dehydration of samples through a graded series of IMS (20%-60%) and ethanol (70%-100%). Samples were left in 100% ethanol and the ethanol was allowed to evaporate overnight. Dried cellularised and acellular samples were mounted on stubs, carbon coated (Agar Auto Sputter Coater, Agar Scientific) and viewed under the SEM microscope (FEI Inspect F, Oxford Instruments, Oxford, UK).

[0225] Results showed cells integrated with the CaP-SM scaffolds in both standard and osteogenic culture conditions (FIG. 19). Cells can be seen forming a layer of interconnected cells which in some areas is embedded within the scaffolds. Some cytoplasmic processes are seen on all samples. These results demonstrate the good cellular properties of CaP-SM scaffolds. [0226] i) Osteogenic Differentiation

[0227] Scaffolds (Smart Matrix used as control, CaP-SM 1 and CaP-SM 2) were cut into 5 mm5 mm square pieces, sterilised, seeded with 110 MC3T3-E1 and cultured in both standard and osteogenic media as already explained.

[0228] Deposition of non-collagenous matrix was assessed by osteopontin immunostaining of paraffin embedded sections. Briefly, antigen retrieval was performed by heat mediated antigen retrieval using sodium citrate buffer at pH 6. After antigen retrieval the sections were blocked using 5% bovine serum albumin at 35 C. for 1 h and stained with primary anti-osteopontin antibody (1:50, abcam ab8448) at 4 C. overnight. Following this, the sections were stained using the alexa fluor 546 secondary antibody for 2 h at room temperature. The sections were then washed and mounted with DAPI based mounting media, cover slipped and visualised using Leica DM IRE2 confocal microscope.

[0229] Mineralisation was assessed by Von Kossa staining as already explained.

[0230] Immunostaining of osteopontin, a protein present in the extracellular matrix of bone, showed that MC3T3-E1 differentiated down the osteogenic pathway in CaP-SM 1 and CaP-SM 2 scaffolds (FIG. 20, where red fluorescence indicates presence of osteopontin; although this may not be readily observed in black and white versions of the figure). Greater differentiation was observed in samples cultured under osteogenic conditions as expected. Von Kossa staining showed extensive mineral deposition in CaP-SM seeded scaffolds in comparison to the Smart Matrix control (FIG. 21).

[0231] These results show that the CaP mineral deposits present in the CaP-SM scaffolds promote differentiation of osteoprogenitor cells down the osteogenic pathway. [0232] j) Angiogenesis

[0233] Proangiogenic; potential of the scaffolds was assessed using an ex ovo chorionic allantoic membrane (CAM) assay. Scaffolds (Smart Matrix used as control, CaP-SM 1 and CaP-SM 2) were cut into 5 mm5 mm square pieces, sterilised with 70% IMS and washed three times with PBS. A negative and a positive control were run alongside the scaffolds: filter paper soaked in either PBS (negative) or 10 ng/ml of vascular endothelial growth factor (VEGF) solution (positive). Fertile chicken eggs were incubated at 37.5 C., 3% CO.sub.2 and humidity was maintained between 35%-45%. At day 3 post-incubation, the embryos were transferred to a shell-less culture system with 75-80% humidity and 37.5 C. incubation temperature. At day 6 of shell-less culture time, scaffolds were applied onto the developing CAMs. Angiogenesis was examined in all the scaffolds macroscopically by taking photos using a stereomicroscope. ImageJ software was used to analyse the macroscopic photos and calculate the percentage of vascular area for each scaffold.

[0234] Results from the CAM assay (FIG. 22) showed that CaP-SM 1 and CaP-SM 2 scaffolds had very similar pro-angiogenic potential to Smart Matrix scaffolds. Smart Matrix has been shown to be pro-angiogenic in vivo, therefore results suggest that CaP-SM 1 and CaP-SM 2 scaffolds would retain this pro-angiogenic potential after addition of a CaP mineral phase. REFERENCES

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