TISSUE SCAFFOLD

Abstract

Tissue scaffold matrices, and methods of their use, are described. The matrices comprise an enzyme that is able to convert a substrate to release hydrogen peroxide and a substrate for the enzyme. The matrices may be impregnated with cells, such as stem cells. Also described are cell cultures, and methods for proliferating and/or differentiating cells.

Claims

1. A matrix for use as a tissue scaffold, comprising a purified enzyme that is able to convert a substrate to release hydrogen peroxide and a substance that includes a substrate for the enzyme.

2. A matrix according to claim 1, comprising one or more fibers.

3. A matrix according to claim 2, comprising one or more nanofibers.

4. A matrix according to any preceding claim, comprising a polymer.

5. A matrix according to claim 4, wherein the polymer is polycaprolactone.

6. A matrix according to any preceding claim, formed by electrospinning.

7. A matrix according to any preceding claim, wherein the enzyme is at least 95% pure or is pharmaceutical grade.

8. A matrix according to any preceding claim, wherein the matrix does not comprise sufficient free water to allow the enzyme to convert the substrate.

9. A matrix according to any preceding claim, wherein the enzyme is an oxidoreductase enzyme, preferably wherein the enzyme is glucose oxidase.

10. A matrix according to any preceding claim, wherein the substance comprises a purified substrate for the enzyme.

11. A matrix according to claim 10, wherein the substrate is at least 95% pure or is pharmaceutical grade.

12. A matrix according to any of claims 10 to 11, wherein the substance comprises a solute with a solubility greater than or equal to 100 g/100 g water at 20 C. and 1 atm, preferably greater than or equal to 200 g/100 g water at 20 C. and 1 atm, more preferably greater than or equal to 300 g/100 g water at 20 C. and 1 atm.

13. A matrix according to claim 12, wherein the solute is a sugar or sugar derivative, preferably wherein the solute is fructose,

14. A matrix according to claim 12 or claim 13, wherein the solute is a purified solute, preferably at least 95% pure or pharmaceutical grade.

15. A matrix according to any of claims 1 to 9, wherein the substance is, or comprises, an unrefined natural substance.

16. A matrix according to any preceding claim, wherein the substance is or comprises honey.

17. A matrix according to any preceding claim which is sterile.

18. A matrix according to any preceding claim which is bioabsorbable or biodegradable.

19. A matrix according to claim 2 or any claim dependent on claim 2 which has a fiber diameter range within 10 to 500 nm, 50 to 300 nm or 100 to 250 nm.

20. A matrix according to claim 2 or any claim dependent on claim 2, with a mean fiber diameter of 10 to 500 nm, 50 to 300 nm, 100 to 250 nm, or 100 to 200 nm.

21. A matrix according to claim 2 or any claim dependent on claim 2, with a mean pore size of 2 m to 1000 m, 2 m to 500 m, 2 m to 250 m or 2 m to 50 m.

22. A matrix according to claim 2, or any claim dependent on claim 2, with a mean pore size of 5 m to 1000 m, 5 m to 500 m, 5 m to 250 m, 5 m to 100 m, or 5 m to 50 M.

23. A matrix according to claim 2 or any claim dependent on claim 2 with a mean pore size of 10 m to 1000 m, 10 m to 500 m, 10 m to 250 m, 10 m to 100 m, or 10 to 50 m.

24. A matrix according to claim 2 or any claim dependent on claim 2, with a mean pore size of 50 m to 1000 m, 50 m to 500 m or 50 m to 250 m.

25. A matrix according to claim 2, or any claim dependent on claim 2, with a mean pore size of 100 m to 1000 m, 100 m to 500 m, or 100 m to 250 m.

26. A matrix according to claim 2 or any claim dependent on claim 2, with a mean pore size of 200 m to 1000 m or 200 m to 500 m.

27. A matrix according to any preceding claim with a porosity of at least 40%, at least 50%, at least 80%, at least 85%, at least 90% or at least 95%.

28. A matrix according to any preceding claim, which comprises substantially no hydrogen peroxide, or no detectable hydrogen peroxide.

29. A matrix according to any preceding claim, which comprises no added peroxidase, substantially no peroxidase, or is essentially free of peroxidase.

30. A matrix according to any preceding claim, which comprises no added zinc oxide, substantially no zinc oxide, or is essentially free of zinc oxide.

31. A matrix according to any preceding claim, in combination with at least one tissue cell.

32. A matrix according to claim 31, impregnated or seeded with the at least one cell,

33. A matrix according to claim 31 or claim 32, wherein the at least one cell comprises a skin cell.

34. A matrix according to any of claims 31 to 33, wherein the at least one cell is a stem cell.

35. A matrix according to any of claims 31 to 34, wherein the at least one cell is a human adipose-derived stem cell (hADSC).

36. A matrix according to any preceding claim with a water activity (a.sub.w) of 0.8 or less,

37. A matrix according to claim 36, with a water activity of 0.2 to 0.8, preferably 0.3 to 0.7.

38. A matrix according to any preceding claim that does not comprise an unrefined natural substance.

39. A matrix according to any preceding claim, which does not comprise honey.

40. A matrix according to any preceding claim, which is pharmaceutical grade, or wherein its components are pharmaceutical grade.

41. A method of repairing damaged tissue in a subject comprising administering a matrix as defined in any of claims 1 to 40, to the damaged tissue.

42. A method according to claim 41, wherein the matrix is implanted into the damaged tissue.

43. A method according to claim 42, wherein the damaged tissue is damaged skin.

44. A method according to any of claims 41 to 43, wherein following administration of the matrix to the damaged tissue, the matrix is not manually removed or replaced.

45. A matrix as defined in any of claims 1 to 40, for use in repairing damaged tissue in a subject.

46. A matrix for use according to claim 45, comprising implanting the matrix into the damaged tissue.

47. A matrix for use according to claim 45 or claim 46, wherein the matrix is not manually removed or replaced.

48. A method comprising contacting cells with a matrix, the matrix comprising an enzyme that is able to convert a substrate to release hydrogen peroxide and a substance that includes a substrate for the enzyme.

49. A method according to claim 48, comprising seeding or impregnating the cells into the matrix.

50. A method according to claim 48 or claim 49, comprising contacting the cells with the matrix in vitro or ex vivo.

51. A method according to any of claims 48 to 50, comprising incubating the cells and the matrix in a nutrient medium, optionally following contacting the cells with the matrix.

52. A method according to any of claims 48 to 49, comprising contacting the cells with the matrix in vivo.

53. Use of a matrix as a tissue or cell scaffold, the matrix comprising an enzyme that is able to convert a substrate to release hydrogen peroxide and a substance that includes a substrate for the enzyme.

54. A matrix for use as a tissue scaffold in repairing damaged tissue in a subject, the matrix comprising a purified enzyme that is able to convert a substrate to release hydrogen peroxide and a substance that includes a substrate for the enzyme.

55. A cell culture or tissue culture comprising a matrix, the matrix comprising an enzyme that is able to convert a substrate to release hydrogen peroxide and a substance that includes a substrate for the enzyme.

56. A method of proliferating and/or differentiating cells comprising contacting cells with a matrix, the matrix comprising an enzyme that is able to convert a substrate to release hydrogen peroxide and a substance that includes a substrate for the enzyme.

57. A method according to claim 56, wherein the cells are contacted with the matrix ex vivo or in vitro.

58. A method according to claim 56 or claim 57, comprising contacting the cells and the matrix with a nutrient medium.

59. A method according to any of claims 56 to 58, comprising incubating.

60. A method according to any of claims 48 to 52, or 56 to 59, wherein the enzyme is a purified enzyme.

61. A method according to any of claims 48 to 52, or 56 to 60, wherein the substrate is a purified substrate.

62. An implant or prosthesis comprising a matrix, the matrix comprising an enzyme that is able to convert a substrate to release hydrogen peroxide and a substance that includes a substrate for the enzyme.

63. A cell culture, implant or prosthesis according to claim 55 or 62, wherein the enzyme is a purified enzyme.

64. A cell culture, implant or prosthesis according to any of claim 55, 62 or 63, wherein the substrate is a purified substrate.

Description

[0214] Embodiments of the invention are now described, by way of example only, with reference to the accompanying drawings in which:

[0215] FIG. 1 shows SEM images of meshes a) 0% SurgihoneyRO; b) 10% SurgihoneyRO; c) 20% SurgihoneyRO and d) 30% SurgihoneyRO, with corresponding insets of fibre distribution; e) Fibre diameter; and f) length;

[0216] FIG. 2 shows water contact angle: a) Images of water droplet on mesh surfaces at 0 and 50 s. b) Contact angle measurements at 0 s and 50 s (*p<0.1);

[0217] FIG. 3 shows a) Cell viability with Live/Dead staining at day 1 and 14 (scale bar 100 m.sup.2); b) Cell proliferation with Alamar Blue assay at day 1, 3, 7, and 14 with NFI;

[0218] FIG. 4 shows a comparison of viscosities of solutions used to make meshes, as a function of shear rate (1/s);

[0219] FIG. 5 shows SEM images of electrospun meshes a) pure PCL, b) 20% SurgihoneyRO, c) 30% SurgihoneyRO and d) mean fiber diameter-honey concentration;

[0220] FIG. 6 shows a cell proliferation as a function of the fluorescence intensity on meshes;

[0221] FIG. 7 is a graph showing the effect of compositions of the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) on the growth of planktonic MRSA, compared to SurgihoneyRO, at various concentrations;

[0222] FIG. 8 is a graph showing the effect of sterile and non-sterile compositions of the invention comprising glucose, glucose oxidase and fructose (buffered at pH 4.03) on the growth of planktonic MRSA, at various concentrations;

[0223] FIG. 9 is a graph showing the effect of sterile and non-sterile compositions of the invention comprising glucose, glucose oxidase and fructose (unbuffered) on the growth of planktonic MRSA, at various concentrations;

[0224] FIG. 10 is a graph showing the effect of sterile and non-sterile compositions of the invention comprising glucose, glucose oxidase and fructose (buffered at pH 7.04) on the growth of planktonic MRSA, at various concentrations;

[0225] FIG. 11 is a table showing the effect of sterile and non-sterile compositions of the invention comprising glucose, glucose oxidase and fructose, on the MIC and MBC of planktonic MRSA, at various concentrations;

[0226] FIG. 12 shows the effect of compositions of the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) on the growth of planktonic MRSA, compared to SurgihoneyRO, at various concentrations;

[0227] FIG. 13 shows the effect of SyntheticRO on the MIC and MBC of planktonic MRSA, compared to SurgihoneyRO, at various concentrations;

[0228] FIG. 14 shows the effect of SyntheticRO comprising glucose, glucose oxidase and fructose (SyntheticRO) on the growth of planktonic MSSA isolate;

[0229] FIG. 15 (a and b) compares SyntheticRO with SurgihoneyRO using planktonic MRSA and MSSA, and FIG. 15 c is a table showing the MICs of a composition of the invention compared to SurgihoneyRO. Planktonic MRSA and MSSA in vitro cultures were grown in the presence of the respective compositions for 18 hours, then the absorbance (OD.sub.595) measured and compared to untreated cultures (n=6);

[0230] FIG. 16 shows the results of an Alkaline Phosphatase Activity (ALP) assay following addition of hADSCs to meshes containing SurgihoneyRO

[0231] FIG. 17 shows the results of an Alazarin Red assay following addition of hADSCs to meshes containing SurgihoneyRO;

[0232] FIG. 18 shows a SEM image of a matrix containing RO100 with PCL (20% RO100);

[0233] FIG. 19 shows the results of an Alamar Blue assay to assess cell viability on matrices containing RO100 and PCL, the matrices comprising different concentrations of RO100; and.

[0234] FIG. 20 shows a prosthetic hip joint coated with a PCL/RO100 mesh produced using electrospinning.

EXAMPLE 1

Materials and Methods

[0235] PCL (M.sub.w 50,000 Da, CAPA 6500, Perstorp Caprolactones, UK) and SurgihoneyRO (Matoke Holdings, UK) meshes were produced using a solution electrospinning system (Profector, Spraybase, Ireland) consisting of high voltage power supply (from 0 kV to 30 kV), software to control a syringe pump system, stainless steel collector and stainless steel emitter*needle) with diameter 1 mm. Acetic acid (Fisher Scientific, UK) was used as a solvent to produce a range of PCL/SH concentrations whilst keeping the processing parameters constant (Table 1). All meshes were vacuumed dried for 24 hr to evaporate acetic acid.

TABLE-US-00001 TABLE 1 Material and processing conditions Total Distance concentration Flow between Honey PC Acetic of the Rate needle and Concentration L SurgihoneyRO Acid mixture Voltage (mL/ collector (%) (g) (g) (mL) (%) (kV) min) (mm) 0 2 0 10 20 17 0.1 187 10 1.8 0.2 10 20 17 0.1 187 20 1.6 0.4 10 20 17 0.1 187 30 1.4 0.6 10 20 17 0.1 187

[0236] Meshes were characterised using scanning electron microscopy (SEM, Hitachi S-3000N, Japan) at an accelerating voltage of 15 kV. Samples were coated with platinum. The images were analysed using Fiji software with the DiameterJ plugin to assess fibre diameter and length [12].

[0237] The wettability of meshes (n=5) was determined through static contact angle measurement (KSV Cam 200, Finland). Images were obtained at 0 and 50 s after droplet formation and subsequently analysed using the Sessile drop technique.

[0238] In vitro biological characterisation of the meshes was performed using hADSCs (STEMPRO, Thermo Fisher Scientific, USA). Cells were cultured with MesenPRO RS media containing 2% (v/v) growth supplement, 1% (v/v) glutamine, and 1% (v/v) penicillin/streptomycin until 80% confluence and harvested by the use of 0.05% trypsin-EDTA solution (Thermo Fisher Scientific, USA) at passage 7. Prior to cell seeding the meshes were sterilised using 80% ethanol for 2 hr and then dried overnight in a sterile laminar flow cabinet. 50,000 cells in 150 L of media were seeded onto each mesh and incubated in a cell culture incubator (37 C., 5% CO.sub.2, and 95% humidity) for 4 hr to allow cell attachment, before the addition of 450 L fresh media. Cell proliferation was assessed at day 1, 3, 7, and 14 after cell seeding, using the resazurin assay (Alamar Blue) (Sigma-Aldrich, UK). On day 1 all samples (n=7) were transferred to a new 24-well plate to enable quantification of cell attachment and prevent unattached cells from influencing the result. Tissue culture plastic (TCP) was used as a control. At each time point, a 10% by volume (60 L) of resazurin solution (0.01% (v/v)) was added to each sample and incubated for 4 hours. After incubation, 150 L of each sample was transferred to a 96-well plate and the fluorescence intensity was measured (540 nm excitation/590 nm emission wavelength) with a plate reader (infinite 200, Tecan, Switzerland). Samples were washed twice in sterile PBS to remove the resazurin solution before the addition of fresh media. Cell culture media was changed every 3 days.

[0239] Cell viability was assessed using a Live/Dead Assay Kit (ThermoFisher Scientific, UK) at day 1 and day 14 according to the manufacturer's instructions. Cell culture media was removed from the samples (n=1) and TCP control and were washed with PBS twice before adding 500 L of calcein-AM and EthD-1, 2 m2 and 4 m.sup.2, respectively, PBS solution. The samples were then incubated for 25 min. Meshes were imaged with an inverted fluorescence microscope (Leica DMI6000 B, Leica Microsystems, Germany).

[0240] Results and Discussion

[0241] SEM images demonstrate the ability to successfully electrospin both PCL and POLISH meshes with nanoscale morphology mimicking the native ECM (FIG. 1). A distribution of fibre diameters ranging from 100-250 nm was observed (FIG. 1a-d inset).

[0242] Increasing the concentration of SH results in a decreasing fibre diameter (FIG. 1e). The average fibre diameter for 0, 10, 20, and 30% meshes are 170, 165, 144, and 136 nm, respectively. However, the fibre length increases with increasing SH concentration due to thinner fibre formation (FIG. 1f). The trend of increasing fibre length and decreasing diameter with increasing SH concentration is potentially related with a decrease in solution viscosity attributed to the reduced PCL content and higher SH concentration of the solution.

[0243] The wettability of the meshes as measured by water contact angle show that at 0 s after droplet formation all meshes present a hydrophobic surface with 0% having the highest contact angle, 124.31, whilst 30% is the lowest (FIG. 2). The contact angle decreases with increasing concentration of SH at both 0 and 50 s. After 50 s meshes containing 30% of SH presents a contact angle of 86.8. As a result, the addition of SH enhances the hydrophilicity of the electrospun meshes.

[0244] Cell seeding efficiency and the cell viability were calculated on day 1. The cell seeding efficiency was over 65% for all SH meshes when compared to the TCP control whilst attachment on PCL was lower. Cell viability, as measured by Live/Dead imaging, shows that approximately 95% of cells were alive (green) on the meshes on day 1 and by day 14 high cell viability was maintained with only few dead cells (red) observed, most likely due to the high cell density after two weeks of proliferation (FIG. 3a). These results indicate that cells are viable on the meshes containing SH.

[0245] All meshes supported cell proliferation as measured by Alamar Blue with a trend of increasing proliferation with higher SH concentration (FIG. 3b). After sterilisation all meshes contracted at different ratios, therefore, the diameter of all meshes was measured with a calliper and the fluorescence intensity was normalised to the area including for the TCP control. The normalised fluorescence intensity (NFI) of all meshes was comparable or greater than the TCP control by day 14. These results illustrate that the cells can attach and proliferate in the meshes. The trend of higher cell proliferation in SH meshes is potentially due to the more hydrophilic surface which allows improved cell spreading and serum proteins from the media attaching in the correct conformation. Furthermore, the SH may possibly be a source of nutrients for proliferating cells.

[0246] Conclusion

[0247] This study demonstrates the successful electrospinning of PCL meshes containing different concentrations of SurgihoneyRO. The meshes exhibit nanoscale features resembling the ECM which show promising biological results with high cell viability and proliferation on all meshes. Subsequently, the meshes demonstrate suitability for tissue engineering applications,

EXAMPLE 2

[0248] Electrospun meshes were produced in a similar manner as in Example 1 but with the following parameters.

TABLE-US-00002 Distance Honey between Con- needle cen- Weight Flow and tration of Weight of Voltage Rate collector (%) PCL (g) Honey(g) (kV) (ml/min) (mm) 0 2 0 18 2 165 20 1.6 0.4 18 2 165 30 1.4 0.6 18 2 165

[0249] The viscosity of the prepared solutions was measured using the HR-2 Rheometer (TA Instruments, Elstree, UK). Viscosities were measured in triplicate for each concentration at room temperature with 0.5 mm zero gap. Results were obtained via the Trios Software.

[0250] Raw SurgihoneyRO samples were tested against the bacterium S. aureus using the minimum inhibitory concentration (MIC) method. MIC tests, also called basic microdilution method, determine the lowest concentration of a chemical compound that prevents the growth of a bacterium.

[0251] Scanning electron microscopy (SEM) was used to assess the morphology structure of the electrospun meshes. Meshes were coated with platinum sputtering during 40 seconds with the Cressington Sputter Coater 108 Auto (Watford, UK). High resolution images were taken using a HITACHI S-3000N (HITACHI, UK) electron microscope at an accelerating voltage of 15 kV. Obtained images were analysed using the DiameterJ software to determine the fibre diameter.

[0252] Electrospun meshes were also biologically assessed in terms of cell proliferation using human adipose-derived mesenchymal stem cells (hADMSC) (StemPro, Thermo Fisher Scientific, UK). Five meshes were considered to determine the effect of each honey concentration on cell proliferation. Cell proliferation was evaluated using the AlamarBlue assay kit (Sigma-Aldrich, UK) according to suppliers' protocol. AlamarBlue reagent includes resazurin the active ingredient of AlamarBlue reagent which is non-toxic and virtually non-fluorescent. When this reagent matched with living cells, it is reduced to resorufin which is a highly fluorescent molecule. Thus, cell proliferation can be quantitatively assessed. Cell proliferation was determined at day 1, 3, 5, 7 and 14. TCP was used as a control.

[0253] Antimicrobial Sensitivity Testing

[0254] Antibacterial properties of SurgihoneyRO were examined against S. aureus. The minimum inhibitory concentration of the SurgihoneyRO was about 6.25% which shows that it is able to inhibit the growth of the bacterium.

[0255] Rheological Behaviour of the PCL-SurgihoneyRO Solutions

[0256] FIG. 4 presents the variation of the viscosity of the PCL-SurgihoneyRO solutions with acetic acid as a function of shear rate.

[0257] Results show that the viscosity decreases by increasing the concentration of SurgihoneyRO and decreasing PCL concentration in the solutions. For low shear rates (0.39 s.sup.1), the viscosity decreases by increasing the shear rate (shear thinning behaviour). For high shear rates the viscosity remains constant with the increase of the shear rate (Newtonian behaviour). In this last regime, the viscosity of the PCL solution is approximately 1.34 Pa.Math.s while the viscosity of PCL/SurgihoneyRO (30%) is approximately 0.42 Pa.Math.s. The initial viscosity of PCL solution is around 10.74 Pa.Math.s and PCL/SurgihoneyRO (30%) is approximately 1.55 Pa.Math.s.

[0258] Morphological Analysis of the Meshes

[0259] SEM images of nonwoven meshes were taken with 7500 magnification at 5 m.sup.2 scale (FIG. 5). Nonwoven PCL and PCL-SurgihoneyRO meshes exhibit a distribution of fibre diameters ranging from 100 nm to 250 nm. Meshes produced with different concentrations of SurgihoneyRO show similar fibre diameter distribution trend. However, the meshes have different fibre diameter values according to the concentration of the SurgihoneyRO. The fibre diameter decreases with increasing the amount of SurgihoneyRO in meshes as follows: 175 nm, 141 nm, and 136 nm for meshes containing 0%, 20% and 30% SurgihoneyRO, respectively (FIG. 5d).

[0260] Biological Results

[0261] Cell proliferation tests were performed over 14 days. Following an initial seeding density of 50,000 cells per well, all meshes presented an increase in fluorescence intensity at each time point up to 14 days in culture media (FIG. 6). The fluorescence intensity of cells in the treated well plate as positive controls showed an increase as well. Meshes without SurgihoneyRO exhibit high cell proliferation up to day 7. However, at day 14 the meshes containing 30% of SurgihoneyRO present the highest fluorescence intensity. Additionally, pure PCL (not containing SurgihoneyRO) meshes have lower fluorescence intensity than the meshes containing SurgihoneyRO. These results show that the fabrication of electrospun meshes with SurgihoneyRO enhances the cell proliferation. Moreover, it also shows that acetic acid does not affect cell proliferation negatively. This means that SurgihoneyRO provides a suitable environment for cell proliferation and adding SurgihoneyRO into meshes does not have a negative effect for cell proliferation.

[0262] Conclusions

[0263] The raw SurgihoneyRO has a good antibacterial property against the bacteria of S. aureus which are commonly found on a skin wound, at the concentration of 6.25%. PCL and SurgihoneyRO mixture are able to be spun together. Therefore, these antibacterial properties show promising application in tissue engineering applications and utilisation in electrospun meshes. Composite PCL-SurgihoneyRO meshes have been fabricated using solution electrospinning process. Meshes have smaller fibre diameter size range from 100 to 250 nm, mimicking the scale of the extracellular matrix (ECM), which means they are suitable for wound dressings. On the other hand, increasing the SurgihoneyRO concentration in the meshes leads to decreasing of fibre diameter, and also the same effect observed for the viscosity. Biological tests using hADSC show that all produced meshes allow cell attachment and proliferation. Moreover, results show at day 14, better results were obtained for meshes containing 30% SurgihoneyRO.

EXAMPLE 3SYNTHETIC HONEY COMPOSITIONS (SYNTHETICRO)

[0264] Samples with batch number RO contain no glucose oxidase.

[0265] Samples with batch number RO1 contain 50 ppm glucose oxidase.

[0266] Samples with batch number RO2 contain 1000 ppm glucose oxidase.

[0267] A. pH 4.03 buffered samples

[0268] A1. Batch no NB01p43RO [0269] Non sterile

TABLE-US-00003 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH 17.0% buffer pH 4.03

[0270] Description

[0271] Non sterile base buffered saccharide solution.

[0272] A2. Batch no NB01p43RO [0273] Sterile

TABLE-US-00004 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH 17.0% buffer pH 4.03

[0274] Description Sterile base buffered saccharide solution

[0275] A3. Batch no NB01p44RO1 [0276] Non sterile

TABLE-US-00005 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH 17.0% buffer pH 4.03

[0277] Description

[0278] Non sterile base buffered RO1 saccharide solution.

[0279] A4, Batch no NB01p44RO1 [0280] Sterile

TABLE-US-00006 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

[0281] Description

[0282] Sterile base buffered RO1 saccharide solution

[0283] A5, Batch no NB01p44RO2 [0284] Non sterile

TABLE-US-00007 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

[0285] Description

[0286] Non sterile base buffered RO2 saccharide solution,

[0287] A6, Batch no NB01p43RO2 [0288] Sterile

TABLE-US-00008 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0% GOX enzyme N/A

[0289] Description Sterile base buffered RO2 saccharide solution

[0290] B. Unbuffered Samples

[0291] B1. Batch no NB01p51RO [0292] Non sterile

TABLE-US-00009 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

[0293] Description

[0294] Non sterile base buffered saccharide solution.

[0295] B2. Batch no NB01p51RO [0296] Sterile

TABLE-US-00010 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

[0297] Description Sterile base buffered saccharide solution

[0298] B3. Batch no NB01p51RO1 [0299] Non sterile

TABLE-US-00011 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

[0300] Description

[0301] Non sterile base buffered RO1 saccharide solution.

[0302] B4. Batch no NB01p51RO1 [0303] Sterile

TABLE-US-00012 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

[0304] Description

[0305] Sterile base buffered RO1 saccharide solution

[0306] B5. Batch no NB01p51RO2 [0307] Non sterile

TABLE-US-00013 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

[0308] Description

[0309] Non sterile base buffered RO2 saccharide solution

[0310] B6. Batch no NB01p51RO2 [0311] Sterile

TABLE-US-00014 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

[0312] Description [0313] Sterile base buffered RO2 saccharide solution

[0314] C. pH 7.04 buffered samples

[0315] C1. Batch no NB01p57RO [0316] Non sterile

TABLE-US-00015 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

[0317] Description

[0318] Non sterile base buffered saccharide solution.

[0319] C2. Batch no NB01p57RO [0320] Sterile

TABLE-US-00016 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

[0321] Description

[0322] Sterile base buffered saccharide solution

[0323] C3. Batch no NB01p57RO1 [0324] Non sterile

TABLE-US-00017 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

[0325] Description

[0326] Non sterile base buffered RO1 saccharide solution.

[0327] C4. Batch no NB01p57RO1 [0328] Sterile

TABLE-US-00018 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

[0329] Description

[0330] Sterile base buffered RO1 saccharide solution

[0331] C5. Batch no NB01p57RO2 [0332] Non sterile

TABLE-US-00019 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

[0333] Description

[0334] Non sterile base buffered RO2 saccharide solution.

[0335] C6. Batch no NB01p57RO2

TABLE-US-00020 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

[0336] Description

[0337] Sterile base buffered RO2 saccharide solution

EXAMPLE 4EFFICACY OF SYNTHETIC HONEY COMPOSITIONS AGAINST PLANKTONIC MRSA

[0338] MIC and MBC were assessed for the RO1 samples (containing 50 ppm glucose oxidase) and compared to SurgihoneyRO (also containing 50 ppm glucose oxidase). See Andrews J. M. Journal of Antimicrobial Chemotherapy (2001) 48, suppl. S1, 5-16.

[0339] The results are shown in FIGS. 7 to 11.

[0340] The results show that, like SurgihoneyRO, synthetic compositions containing glucose, glucose oxidase and fructose are able to inhibit microbial growth.

[0341] Out of all of synthetic compositions, the synthetic composition buffered at pH7.04 had the most effective MIC. Sterilised compositions were more effective than non-sterilised compositions, and synthetic composition buffered at pH7.04 synthetic had the most effective MBC when compared to other synthetic compositions and even when compared to SurgihoneyRO.

[0342] FIGS. 12 (a to d) and 13 show MIC and MBC results including SurgihoneyRO2 samples and synthetic RO2 samples.

[0343] pH 7.04 formulations were tested against a planktonic MSSA isolate. FIG. 14 shows the results obtained.

[0344] The synthetic RO2 composition was selected for further investigation. FIG. 15 (a, b and c) show SyntheticRO (RO2; pH7.04) compared to SurgihoneyRO using planktonic phenotype. RO indicates a product lacking enzyme activity.

EXAMPLE 5CELL EXPERIMENTS

[0345] In vitro biological characterisation of the meshes was performed using hADSCs (STEMPRO, Thermo Fisher Scientific, USA). Cells were cultured with MesenPRO RS media containing 2% (v/v) growth supplement, 1% (v/v) glutamine, and 1% (v/v) penicillin/streptomycin until 80% confluence and harvested by the use of 0.05% trypsin-EDTA solution (Thermo Fisher Scientific, USA) at passage 7. Prior to cell seeding the meshes were sterilised using 80% ethanol for 2 hr and then dried overnight in a sterile laminar flow cabinet. 15,000 cells in 150 L of media were seeded onto each mesh and incubated in a cell culture incubator (37 C., 5% CO.sub.2, and 95% humidity) for 4 hr to allow cell attachment, before the addition of 350 L fresh media.

[0346] Meshes containing SurgihoneyRO and PCL (Surgihoney: 10%, 20% and 30%) were assessed using the following cell assays.

[0347] Alkaline Phosphatase Activity (ALP)

[0348] To investigate the osteogenic differentiation of hADSCs seeded on the meshes, alkaline phosphatase enzyme activity was observed using a colorimetric assay (SensoLYTE Pnpp Alkaline Phosphatase Assay Kit, AnaSpec, Fremont, Calif., USA), using manufacturer's protocol.

[0349] Firstly, solutions were prepared which were used in the experiment. [0350] 1ALP assay buffer solution was prepared from 10ALP assay buffer solution (Manufacturer provided). [0351] 1 ml of 10ALP assay buffer solution mix with 9 ml of deionized water to make 1ALP assay buffer. [0352] 0.2% v/v Triton X-100 was prepared. 20 l was added to 10 ml of 1ALP assay buffer to make 0.2% v/v Triton X-100 [0353] 1. Samples (n=3) were transferred to 24 well plates [0354] 2. Samples were washed twice with ALP dilutions assay buffer. [0355] 3, Samples were transferred from 24-well plates to 1.5 ml Eppendorf tubes. [0356] 4. 250 l 1ALP assay buffer containing 0.2% v/v Triton X-100 was added to each tube. [0357] 5. Each sample was vortexed (in the Eppendorf tubes) for 1 min. [0358] 6. All samples (in the Eppendorf tubes) were centrifuged at 1700g for 15 mins at 4 C. [0359] 7. 50 l supernatants were taken from the each tube and transferred to 96-well plates. [0360] 8. 50 l pNPP (manufacturer provided) was added into supernatants in the 96-well plates and left at room temperature in the dark by covering with aluminium foil for 1 h. [0361] 9. 50 l stop solution (manufacturer provided) was added to into 96 well plate, to form a 150 l solution each well. [0362] 10. Absorbance was measured at 405 nm.

[0363] FIG. 16 shows the results at day 7 and day 14. It is noted that alkaline phosphatase activity is an early marker of bone development.

[0364] Alizarin Red

[0365] To investigate the mineralisation of hADSCs seeded on the meshes, Alizarin red-S(ARS) (Sigma Aldrich, Dorset, UK) assay was used. [0366] 1. 0.2% w/v ARS solution was prepared using ARS powder and distilled water. This solution was covered with foil to protect it from the light. [0367] 2. Meshes were transferred to 24 well plate and then washed with PBS twice. [0368] 3. Meshes were immersed in 10% Formaldehyde solution 15 mins at room temperature. [0369] 4. Formaldehyde solution was removed and samples washed with deionized water three times. [0370] 5. 0.2% ARS staining solution was added to meshes until covering the meshes. [0371] 6. 24-well plates covered were covered with foil and left for 40 mins at the room temperature in the dark. [0372] 7. Samples were washed with deionized water 5 times (each times after a 5 min wait). [0373] 8. Meshes were transferred to 1.5 ml Eppendorf tubes and 800 l 10% Acetic Acid solution was added into these tubes, and the tubes were shaken gently at room temperature for 30 mins. [0374] 9. The solutions were transferred to new Eppendorf tubes. [0375] 10. The solutions in new tubes were vortexed for 30 seconds each. [0376] 11. The solutions were heated at 85 C. for 10 mins. To avoid evaporation, the tubes were sealed with film. [0377] 12. The tubes were kept in the freezer for 5 mins to cool down. [0378] 13. The tubes were centrifuged at 1700g for 15 mins. [0379] 14. After centrifugation, 150 l supernatants transferred to 96-well plates and absorbance was measured at 405 nm.

[0380] FIG. 17 shows results at day 7 and day 14. Alizarin Red stains calcium which is formed during osteogenic differentiation and bone development. All samples show calcium formation.

EXAMPLE 7MESH PRODUCTION FOR NON-HONEY-BASED, SYNTHETIC COMPOSITION

[0381] This example utilised a synthetic composition (referred to as RO100), comprising glucose (31%, by weight), fructose (52%, by weight). Water (17%, by weight) and glucose oxidase (0.5% by weight).

[0382] RO100-based matrices were formulated by electrospinning compositions containing PCL, R0100 and acetic acid. The relative amounts of RO100 and PCL were varied (10%, 20% and 30% RO100).

[0383] It was found that RO100-based matrices could be formed when using, for example, a distance between needle and collector of 15 cm, a flow rate of 0.1 ml/minute, a voltage of 15 kV or 10 kV and a processing time of 20 minutes.

[0384] FIG. 18 shows an SEM image of a 20% RO100 mesh obtained using a voltage of 10 kV.

[0385] The mean fiber diameter for all produced samples was within 180-300 nm, with porosity (measured using DiameterJ software) between 0.46 and 0.59.

EXAMPLE 8CELL VIABILITY TEST

[0386] Proliferation of hADSCs was detected on RO100-based matrices using the AlamarBlue assay (See Examples 1 and 2). The results are shown in FIG. 19. It is noted that over the 14 day period, the increase in stem cell count was higher for both the 10% RO100 matrix (138%) and the 20% RO100 matrix (18%) compared to a matrix comprising PCL but without RO100 (3%).

EXAMPLE 9COATING MEDICAL DEVICES

[0387] FIG. 20 shows a prosthetic hip joint coated with a PCL/RO100 mesh produced by electrospinning a solution comprising PCL, acetic acid and RO100.

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