Dressing

Abstract

The invention relates to a surgical or wound dressing comprising a sheet of gellan gum and an antifibrotic agent. Methods of producing dressings comprising gellan gums and biologically active agents area also produced.

Claims

1. A process for making a surgical, burn, or wound dressing, the process comprising: (a) heating about 2% to about 5% w/w gellan gum in a first aqueous liquid, and optionally about 5% w/w or less of a polymer, to form a liquid gellan gum; (b) cooling the liquid gellan gum; (c) adding one or more bioactive agents to the cooled liquid gellan gum; (d) casting the cooled liquid gellan gum to form a sheet having a thickness of about 0.5 mm to about 5 mm; and (e) drying the sheet; where the sheet is permeable, and where upon rehydration, the sheet is sufficiently transparent to allow a surgical incision, burn, or wound to be viewed.

2. The process of claim 1 further comprising mixing the gellan gum and aqueous liquid at about 80° C. or greater to form the liquid gellan gum.

3. The process of claim 1 wherein the liquid gum is cooled to about 37° C. to about 45° C.

4. The process of claim 1 wherein at least one of the one or more bioactive agents is an antifibrotic agent.

5. The process of claim 4 wherein the antifibrotic agent is added to the cooled liquid gellan gum at about 37° C. to about 45° C.

6. The process of claim 1 further comprising rehydrating the dried sheet with a second aqueous liquid, where the second aqueous liquid optionally comprises one or more bioactive agents.

7. The process of claim 6 further comprising compressing the rehydrated sheet to remove at least a portion of the second aqueous liquid from the sheet; and adding a third aqueous liquid comprising one or more bioactive agents, whereby the one or more bioactive agents are included in sheet.

8. The process of claim 7 wherein at least one of the one or more bioactive agents is selected from the group consisting of antifibrotic agents, pro-VEGF agents, anti-VEGF agents, antibacterial agents, and proteoglycans.

9. The process of claim 1 further comprising adding a cross linking agent to the gellan gum and the first aqueous liquid.

10. The process of claim 9 wherein the crosslinking agent is a cation.

11. The process of claim 1 wherein the additional polymer is gelatin or polyvinyl acetate, or a combination thereof.

12. The process of claim 11 wherein the polyvinyl acetate is partially or fully saponified.

13. The process of claim 1 wherein the dressing is configured to allow fluid to pass into the dressing.

14. The process of claim 1 wherein the dressing is configured to allow fluid to pass through the dressing.

15. A process for making surgical, burn, or wound dressing, the process comprising: (a) heating about 2% to about 5% w/w gellan gum in a first aqueous liquid, and optionally about 5% w/w or less of a polymer, to form a liquid gellan gum; (b) cooling the liquid gellan gum; (c) adding one or more bioactive agents to the cooled liquid gellan gum; (d) casting the cooled liquid gellan gum to form a sheet having a thickness of about 0.5 mm to about 5 mm; and (e) drying the sheet; where the sheet is hydratable, and where upon rehydration, the sheet is sufficiently transparent to allow a surgical incision, burn, or wound to be viewed.

16. The process of claim 15 wherein the liquid gum is cooled to about 37° C. to about 45° C.

17. The process of claim 15 wherein at least one of the one or more bioactive agents is an antifibrotic agent.

18. The process of claim 15 further comprising rehydrating the dried sheet with a second aqueous liquid, where the second aqueous liquid optionally comprises one or more bioactive agents.

19. The process of claim 15 further comprising adding a cross linking agent to the gellan gum and the first aqueous liquid.

20. The process of claim 15 wherein the additional polymer is gelatin or polyvinyl acetate, or a combination thereof.

Description

(1) The invention will now be described by way of example only with reference to the following figures:

(2) FIG. 1 shows a hydrogel dressing sheet manufactured according to the invention.

(3) FIG. 2 shows the effect of a variety of calcium chloride concentrations on the G′ of gellan membranes.

(4) FIG. 3 compares the release of decorin, decorin from gellan membranes and amniotic membranes. FIG. 3a shows the difference between gellan only and gellan cross-linked membranes. FIG. 3b shows the release from amniotic membrane.

(5) FIG. 4 shows an in vitro assessment of gellan membranes and shows the collagen deposition from MFs in the presence of gellan membranes, and, which have been assayed using sirius red.

(6) FIG. 5 shows that decorin significantly reduces the amount of collagen deposited over a 12 day period when compared to non-treated samples.

(7) FIG. 6 shows a schematic diagram of the surface modification of gelatin to allow the attachment of MF cells.

(8) FIG. 7 shows the effect of adding PVA (polyvinyl alcohol) to gellan.

MATERIAL AND METHODS

(9) Industrial grade gellan was obtained from Kelco Limited (Surrey, United Kingdom) under the trade name Gelzan. Calcium chloride, and phosphate buffered saline (Dulbeccos A) were obtained from Sigma Aldridge Company, Dorset, United Kingdom. PETRI dishes, filter paper range (QL100-240 mm) and disposable 10 mm plastic syringes were obtained from Fisher Scientific, Loughborough, United Kingdom. The incubator used by an INCU-LINE, obtained from VWR, Sussex, United Kingdom.

(10) 2% w/w gellan hydrocolloid solution was prepared by weighing 2 grams of gellan powder using a calibrated balance. 98 ml of distilled water was measured and transferred to a glass bottle. Water was placed on a hot plate stirrer at 100° C. and 200 rpm. Gellan powder was added to the stirring water and left for four hours.

(11) A 1% w/w calcium chloride solution was prepared by dissolving 1 gram of calcium chloride in 99 ml of distilled water.

(12) The gellan hydrocolloid was stirred on a hot plate at 90° C. until the gellan became a liquid. This was then reduced to 40° C. 2.5 ml of gellan was syringed into a 55 mm petri dish using a plastic syringe. This was allowed to set for 15 minutes. To dehydrate the membrane sheet, the gel was placed in a 37° C. incubator for 6-8 hours.

(13) To prepare a cross-linked sheet, the gelled gellan hydrocolloid was stirred on a hot plate at 90° C. at 200 rpm, until the gellan became liquid. This was reduced to 40° C. A filter paper was soaked in the calcium chloride solution and placed on the bottom of a petri dish. 2.5 ml of gellan was placed onto the filter paper using a plastic syringe. The gel was allowed to set for 5 minutes. A further filter paper soaked in the calcium chloride solution was placed on top of the set gel and left at room temperature for 15 minutes. The filter papers were then carefully removed and the membrane sheet was transferred into an incubator at 37° C. for 6-8 hours.

(14) In order to rehydrate the sheets, the sheets were soaked in PBS for 2-3 minutes.

(15) FIG. 1 shows an example of the dehydrated sheet.

(16) The Effect of Various Calcium Chloride Concentrations on G′ Gellan Membranes

(17) Gels were prepared with a range of different concentrations of calcium chloride. 276 mM calcium chloride, the gel was stiff, not easily drapable or easy to place around a wound. Gels prepared with 15 mM were shown to be more drapable around a spherical object, such as a marble, but were susceptible to tearing at maximum strain. 10 mM calcium chloride were more fragile and would tear more easily when lifted out of the mould. Therefore 15 mM calcium chloride was the preferred concentration to use. The effects on G′ are shown in FIG. 2.

(18) FIG. 3 shows a comparison of the release of decorin from gellan membranes and amniotic membranes. Gellan-decorin membranes or amniotic membranes were placed in a release medium (Phosphate buffer saline, PBS) and placed in a shaker at 80 rpm at 37° C. Equal amounts of the release medium were taken at specific time intervals and equal amounts of fresh release medium were replaced to maintain a constant volume. The samples were analysed using a human decorin ELISA kit according to manufacturer's instructions

(19) FIG. 4—MFs were cultured in 6-well plates and left to attach overnight. After overnight attachment TGF-β1 was added to the cells to produces excess collagen. Decorin containing membranes were placed on top of the cultures and collagen production was observed using the Sirius Red assay over time.

(20) FIG. 5: For quantifying the amount of Sirius red dye bound to collagen in the cultures the Sirius red was dissolved with 0.1N sodium hydroxide and left on a shaker for 30 min at room temperature. The dissolved dye was measured colourimetrically using a plate reader at 550 nm

(21) FIG. 6: Gellan membranes were produced and cross-linked with 200 mM calcium chloride, and immersed in 10 mM sodium periodate solution for 2 h. The membranes were washed with PBS and further immersed in 10% (w/v) gelatin and 0.5% (v/v) NaBH.sub.4. Furthermore the membranes were soaked overnight in EDC at 4° C.

(22) To determine surface modification, MF cells were left to attach on the surface on membranes for 24 h and cell attachment was assessed using Calcein AM and Propidium Iodide staining.

(23) FIG. 7: Gellan membranes were produced with various amounts of PVA at 10:0, 50:50 and 20:80 (wt %) of gellan:polymer ratios. Tensile testing was carried out using a universal Instron machine to determine the physical properties of the gellan/PVA membranes

(24) Method of Spray Forming a Gel Dressing

(25) 2% w/w of gellan was dissolved in deionised water. A layer of the gellan gum at 60° C. was sprayed onto a mould. After spraying a layer of dextran blue, which was used as a substitute for decorin because it has similar properties and is visible to the naked eye, was sprayed onto the layer of gellan after a 5 second interval to allow the gellan to cool and gel. A 5 second interval was allowed prior to adding another layer of gellan gum and the process repeated 5 times. The whole process took approximately 40 seconds.

(26) It was found that it was possible to form a structure with alternating layers of gellan and dextran blue, which was membrane-like. The advantage of this system is that it allows the material to be rapidly produced. Rastering the spray backwards and forwards across the surface allows a substantially even distribution of the material. Dextran blue was substantially evenly distributed within the gel dressing.

(27) The advantage of this system is that it improves the ability to produce the dressing. It also lends itself to automation in that the spraying of the materials can be readily automated.