Tissue dressing kit

Abstract

A kit comprising a tissue dressing material for being applied in contact with the tissue of a patient and a detachment solvent for removing the tissue dressing material from the tissue. A method of treating a tissue of a patient, the method comprising the steps of: applying a water-soluble tissue dressing material in contact with the patient's tissue; and applying an acidic detachment solvent to the tissue dressing material for removing the tissue dressing material from the tissue. A method of treating a tissue of a patient, the method comprising the steps of: applying a liquid tissue dressing material in contact with the patient's tissue; and applying a detachment solvent to the tissue dressing material for removing the tissue dressing material from the tissue. A method of treating a tissue of a patient, the method comprising the steps of: applying a water-soluble tissue dressing material in contact with the patient's tissue; and allowing the water-soluble tissue dressing material to convert into a form in which it is insoluble in water at neutral pH. And a solid material comprising a polymer salt and glycerol, the glycerol content being at least 10% of the polymer salt content by weight, for use in a method of treating a human or animal tissue.

Claims

1. A method for treating a tissue of a patient, the method comprising: applying a water-soluble solid tissue dressing material or a water soluble gel-like tissue dressing material in contact with the patient's tissue, wherein the tissue dressing material comprises chitosan with a degree of acetylation of less than 2.5%; and allowing the water-soluble solid tissue dressing material or the water soluble gel-like tissue dressing material to convert into a form in which it is insoluble in water at neutral pH.

2. The method of claim 1, comprising: after allowing the water-soluble solid tissue dressing material or the water soluble gel-like tissue dressing material to convert into a form in which it is insoluble in water at neutral pH, applying an acidic detachment solvent for removing tissue dressing material from the tissue to the water-soluble solid tissue dressing material or the water soluble gel-like tissue dressing material by at least partly dissolving the water-soluble solid tissue dressing material or the water soluble gel-like tissue dressing material.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention is illustrated in greater detail with the aid of the following figures:

(2) FIG. 1 shows an .sup.1H NMR spectrum of native chitosan as purchased;

(3) FIG. 2 shows an .sup.1H NMR spectrum of native chitosan essentially deacetylated after further hydrolysis steps;

(4) FIG. 3 illustrates the cell viability of keratinocytes on chitosan materials of various degrees of acetylation, relative to tissue culture polystyrene controls (PS=100%);

(5) FIG. 4 illustrates the controlled dissolution of tissue dressing material according to the invention by applying a gauze soaked with acetate buffered solution;

(6) FIG. 5 shows a tissue dressing comprising a tissue dressing material according to the invention before (5a), during (5b), and after (5c) the application of the detachment solvent;

(7) FIG. 6 schematically illustrates a wound to which a liquid tissue dressing material according to the inventions has been applied;

(8) FIG. 7 schematically illustrates a wound to which a solid tissue dressing material according to the inventions has been applied;

(9) FIG. 8 schematically illustrates a wound to which a non-perforated wound dressing according to the invention has been applied; and

(10) FIG. 9 schematically illustrates a wound to which a perforated wound dressing according to the invention has been applied.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

(11) 1. .sup.1H NMR Spectroscopy

(12) The chitosan used as a starting material in the examples below was obtained in the form of fine flakes from Cognis (Germany). The degree of acetylation (DA) was determined by .sup.1H NMR spectroscopy. FIG. 1 shows an .sup.1H NMR spectrum obtained from this commercially available chitosan. FIG. 2 shows a corresponding .sup.1H NMR spectrum obtained from chitosan deacetylated after further hydrolysis steps applied to the commercial product as described further below. In both cases, chitosan was analyzed in a mixture of 0.25% DCI in D.sub.2O at a chitosan concentration of approximately 0.5% (w/v). The spectra were recorded using a Bruker AC200 spectrometer. NMR chemical shifts (δ, in ppm) were referenced to the signal of HDO (δ=4.8 ppm). The DA, calculated by comparing the integrated area under the peaks associated with H2-H6 of the D-glucosamine subunit with that of the methyl group, was determined as 14.5% for the native chitosan as purchased, and 1.5% for the deacetylated native chitosan.

(13) 2. Synthesis of Low-DA Chitosan

(14) For further hydrolysis, 50 g (grams) of the chitosan flakes as obtained from the supplier Cognis were placed in a glass container, and 500 g of a 45% aqueous sodium hydroxide solution were added. The glass container was well shaken to mix the components, and placed in an oven for 2 hours at 100° C. It was then removed from the oven, and 500 mL (milliliters) of distilled water were added. The mixture was filtered through a glass frit. Then, the chitosan was washed with distilled water until the pH of the filtrate reached 6.5, and dried at 100° C. for 4 h (hours). This hydrolysis treatment was then repeated, resulting in 42 g of deacetylated native chitosan having a DA of 1.5% as determined by 1H NMR spectroscopy.

(15) 3. Cell Viability on Low-DA Chitosan

(16) Human HaCaT keratinocytes were cultured in serum-free medium (Gibco) supplemented with 0.2 ng/mL (nanograms per milliliter) rEGF and 25 μg/mL (micrograms per milliliter) bovine pituitary extract. The calcium concentration was adjusted to 0.02 mM and the pH to 7.2-7.4. Cells were seeded at a density of 1×10.sup.6 cells per 20 mL medium and incubated at 37° C. in air containing 10% CO.sub.2. Cells were passaged once per week, and passages 20-25 were used for analysis.

(17) Chitosan films having DAs of 1.5, 4.0, and 14.5%, respectively, were placed in 24-well cell culture plates, and human HaCaT keratinocytes were seeded at a density of 5×10.sup.4 cells per cm.sup.2 and cultured for 2 days. Cell viability was determined using the MTS assay (Promega). After 4 h of MTS incubation with the cells, the light absorbance at 490 nm was measured by an ELISA plate reader and subtracted from that of the controls (without cells) to yield the corrected absorbance. Five samples of each DA were studied. FIG. 3a shows the relative light absorbencies a at 490 nm (PS=100%) for the three samples and a control using polystyrene (PS).

(18) 4. Preparation of a Solution of the Tissue Dressing Material (Material A)

(19) 7.5 g of the thus obtained native chitosan having a DA of 1.5% were dissolved in 500 mL of a 0.5% aqueous acetic acid by gently shaking for 24 h. A portion of the solution was filtered first through a glass fiber filter (pore size approximately 1 μm), and then through a 0.22 μm filter for sterilization, resulting in a solution of a tissue dressing material essentially consisting entirely of deacetylated native chitosan. Below, the material is referred to as tissue dressing material A.

(20) 5. Preparation of a First Example of a Solid Film-Type Tissue Dressing Material (Material B)

(21) Two portions of 144 mL each of the non-filtered solution of deacetylated native chitosan prepared above were poured into two square-shaped moulds, 24×24 cm.sup.2 (square centimeters) in size, and left in a dust-free environment for drying at room temperature. The resulting film was removed from the first mould, and sterilized using a 10 kGy (kilogray) electron beam. An approximately 80 μm thick transparent film of tissue dressing material essentially consisting entirely of deacetylated native chitosan acetate salt was obtained. Below, the material is referred to as tissue dressing material B.

(22) 6. Preparation of a Second Example of a Solid Film-Type Tissue Dressing Material (Material C)

(23) The dried film from the second mould was placed for 2 hours in a bath containing a solution of 1.5% ammonia in methanol/water 90/10 (v/v). The film was then removed from the bath and dried by storage at room temperature. The film was sterilized using a 10 kGy electron beam. An approximately 80 μm thick transparent film of tissue dressing material essentially consisting entirely of deacetylated native chitosan base was obtained. Below, the material is referred to as tissue dressing material C.

(24) 7. Preparation of a Third Example of a Solid Film-Type Tissue Dressing Material (Material D1)

(25) 144 mL of the filtered solution of deacetylated chitosan prepared as above was poured into a square-shaped mould, 24×24 cm.sup.2 in size, and left in a dust-free environment for drying at room temperature. After 3 days of storage, the resulting film was removed from the mould, transferred in a plastic bag that was then tightly sealed, and sterilized using a 25 kGy (kilogray) electron beam. An approximately 80 μm thick transparent film of tissue dressing material essentially consisting entirely of deacetylated chitosan acetate salt was obtained. Below, the material is referred to as tissue dressing material D1.

(26) 8. Preparation of a Fourth Example of a Slid Flm-Type Tissue Dressing Material (Material D2)

(27) In a slightly modified procedure, 4% (w/w) glycerol was added to the filtered solution of deacetylated chitosan before pouring it into the square-shaped mould. Subsequent treatment as described above for tissue dressing material D1 resulted in a transparent film of tissue dressing material essentially consisting entirely of a mixture of deacetylated chitosan acetate salt and glycerol. Below, the material is referred to as tissue dressing material D2.

(28) 9. Preparation of a Fifth Example of a Solid Film-Type Tissue Dressing Material (Material D3)

(29) In a further modified procedure, the glycerol containing solution of deacetylated chitosan was poured into a square-shaped mould which was covered with a two-layered film consisting of polyurethane/polyethylene (Platilon U073 PE, Epurex, Bomlitz/Germany), with the polyurethane side up and the polyethylene side fixed to the bottom of the mould. Subsequent treatment as described above for tissue dressing material D1 resulted in a transparent film of tissue dressing material essentially consisting entirely of a mixture of deacetylated chitosan acetate salt and glycerol which was attached to the polyurethane/polyethylene support film. Below, the material is referred to as tissue dressing material D3. Upon use, the polyethylene layer is removed. The remaining polyurethane layer is gas-permeable.

(30) 10. Preparation of a Sixth Example of a Solid Film-Type Chitosan (Chitosan D4)

(31) In a slightly modified procedure to the preparation of chitosan film D2, 1% (w/w) glycerol was added to the filtered solution before pouring it into the square-shaped mould. Subsequent treatment as described above for chitosan D1 resulted in a transparent film essentially consisting entirely of a mixture of deacetylated chitosan acetate salt and glycerol. Below, the material is referred to chitosan D4.

(32) 11. Preparation of Two Examples of a Solid Film-Type Tissue Dressing Material with Higher DA (Materials E1 and F1)

(33) Two further examples of tissue dressing materials were produced by the procedure leading to material D1 with the only modification that in one case the hydrolysis step was shortened, leading to a DA of 4% (material E1), and in the other case the hydrolysis step was entirely omitted, leading to a DA of 16% (material F1).

(34) 12. Water Uptake of Tissue Dressing Material C

(35) Tissue dressing material C, produced as described in the above example, was weighted, and then placed in distilled water for 15 min. The weight of the wet film was compared to the weight of the dry film, and the water uptake was determined to be 72% by weight.

(36) 13. Water Uptake of Chitosan D4

(37) Chitosan D4, produced as described in the above example, was weighted, and then placed in distilled water for 60 min. The weight of the wet film was compared to the weight of the dry film, and the water uptake was determined to be 1217% by weight 7 days after film preparation, and 475% by weight 14 days after film preparation.

(38) 14. Application of Tissue Dressing Materials A, B and C

(39) In Table 1 below, the outcomes of treatments of patients with tissue dressing materials A, B and C are detailed. Material A was sprayed directly onto a wound and then left uncovered to allow the solvent to readily evaporate into the air. Materials B and C were applied as small cuts of the film-like material in direct contact with the wound. In the case of material B, the skin was pre-wetted before the application of the material. In all examples, the material was left uncovered after application.

(40) TABLE-US-00001 TABLE 1 Type of chitosan tissue Application of dressing Patient Wound the dressing Outcome A female, cut of 3.5 cm application of skin 49 yrs length and 5 mm appr. 2 mL of incision (years) depth (finger), tissue dressing A completely moderate (one treatment) closed bleeding after 24 h B male, cut of 1.5 cm application of skin 42 yrs length and 3 mm tissue dressing B incision depth (hand), (size 2 × 0.5 cm.sup.2) completely weak bleeding on pre-wetted skin closed (one treatment) after 4 h C male, praeputial application of Wound and 57 yrs inflammation tissue dressing C ulcus (size 1.5 × 1.5 completely cm.sup.2) (treatment healed repeated after 24 after 48 h h)
15. Dissolution of Chitosan Tissue Dressing

(41) Controlled dissolution of tissue dressing materials B and C was tested in dissolution experiments using distilled water, 0.9% aqueous sodium chloride solution, and 0.5% acetic acid/acetate buffered solution, respectively. The pH of the solutions was adjusted to the values indicated in Table 2 using appropriate amounts of 1 N hydrochloric acid or sodium hydroxide solutions. Materials B and C were cut into rectangular samples having dry weights between 5 and 10 mg each. A gauze soaked with a 100-fold per volume excess of the respective solution to the dry weight of the film was applied to each sample film and the time for complete film dissolution was recorded.

(42) TABLE-US-00002 TABLE 2 Material B Material C Material C pH of the Material B (0.9% aqueous (0.9% aqueous (0.5% acetic dissolution (distilled sodium sodium acid/acetate mixture water) chloride) chloride) buffer) 4.0 n.a. n.a. n.d. 0.5 h 4.5 n.a. n.a. n.d. 0.5 h 5.0 n.a. n.a. n.d. .sup. 2 h 5.5 0.1 h 0.5 h n.d. .sup. 4 h n.a. = not analyzed n.d. = no dissolution observed after 24 h

(43) The controlled dissolution experiment with tissue dressing material C and a mixture of 0.5% acetic acid/sodium acetate (right column in Table 2), is illustrated in FIG. 4. The material has been stained by storage in 0.01% aqueous indigocarmine solution for 1 hour for better visualization. Complete dissolution was observed after 30 minutes at pH 4.0 and 4.5, after 2 hours at pH 5.0, and after 4 hours at pH 5.5, respectively.

(44) 16. In situ Conversion of Water-Soluble Tissue Dressing Material into Water-Insoluble Tissue Dressing Material

(45) Samples of tissue dressing materials A, D1, D2, D3, and D4, respectively, were left unsealed on air at room temperature and a humidity of 20-40%. Under these conditions, tissue dressing A was drying to a solid film within several hours. Complete dissolution in distilled water was analyzed at days 3, 7, and 14. Results are summarized in Table 3.

(46) TABLE-US-00003 TABLE 3 Tissue Dressing Day 3 Day 7 Day 14 A soluble insoluble insoluble D1 soluble soluble insoluble D2 soluble insoluble insoluble D3 soluble insoluble insoluble D4 soluble insoluble insoluble

(47) Similarly, conversion of the water-soluble into the water-insoluble form of the wound dressings A, D1, D2, D3, and D4, respectively, was observed after application of the wound dressing on human skin. In the case of D3, the wound dressing was applied to the skin with its chitosan side. Conversion of the water-soluble into the water-insoluble form of the wound dressings A, D1, D2, D3, and D4, respectively, was also observed after alkaline treatment or storage in an alkaline atmosphere.

(48) 17. Dissolution of Tissue Dressing Material with Detachment Solvent

(49) Tissue dressing materials D1, E1 and F1 were dissolved by storage in a 2% acetic acid/acetate buffered solution. The pH of the solutions was adjusted to the values indicated in Table 4 using appropriate amounts of 10% sodium hydroxide solutions. Films D1, E1 and F1 made from chitosans with different degrees of acetylation (DA) were left on air for 14 days for conversion into the water-insoluble form, cut into rectangular samples of 1×1 cm.sup.2 size and stored in approximately 10 mL of the respective solution, and the time for complete film dissolution was recorded.

(50) TABLE-US-00004 TABLE 4 DA = 16% DA = 4% DA = 1.5% pH Time for complete dissolution (min) 4.0 5 10 1 4.5 15 15 2 5.0 30 15 15 5.5 60 60 30 6.0 60 overnight overnight

(51) In another dissolution experiment, a wound dressing material film C (3×1 cm.sup.2) was fixed on the inside of a commercial perforated band-aid (5×2 cm) which was then fixed on a Petri dish. An acetic acid/acetate buffered solution (pH 5.5) was added dropwise through the perforations of the band-aid causing the wound dressing material film to dissolve. The side of the tissue dressing comprising the tissue dressing material film is shown in FIG. 5a before and in FIG. 5c after application of the solution. The application of the solution to the band-aid side of the wound dressing is shown in FIG. 5b.

(52) In FIG. 6, schematically a tissue 1 comprising a wound 2 is shown. For better illustration, FIGS. 5 to 8 are not drawn to scale. The liquid tissue dressing material according to the invention has been applied to the tissue 2 and the constituent water has been allowed to evaporate, leaving behind a film 3 that dresses the tissue 2 including the wound 3. In general, the film 3 is about 10 to 20 μm thick. Advantageously, the film 3 tightly snuggles to the tissue surface 4, including the wound surface 5.

(53) FIG. 7 schematically shows a tissue dressing material in the form of a solid film 6 that is applied to a tissue 1, comprising a wound 2. The solid film is about 80 μm thick. Cavities 7, 8 between the tissue 1 and the tissue dressing material 6 may be filled with water or exudative fluid.

(54) In FIG. 8, a tissue dressing 9 comprising a tissue dressing material 6 of FIG. 7 as a first layer and a silicon film 10 as a second layer is applied to a tissue 1, comprising a wound 2. The silicon film 10 is about 50 μm tick. Again, cavities 7, 8 between the tissue 1 and the tissue dressing material 6 may be filled with water or exudative fluid. Finally, FIG. 9 shows a tissue dressing 11 applied to a tissue 1 comprising a wound 2, the tissue dressing 11 differing from that 9 of FIG. 8 in that the silicon film 10 is perforated to allow an exchange of air between the tissue 1 and the surrounding though the wound dressing material 6. The perforations have a diameter if between 50 and 100 μm.

(55) 18. Preparation of an Example of a Liquid-Type Tissue Dressing Material

(56) 1000 ml of tissue dressing material A was diluted by addition of 1000 mL of sterile distilled water, resulting in a liquid-type tissue dressing material consisting of 0.75% chitosan, 0.25% acetic acid and 99% water. Using a mechanical dispenser, 20 mL of the solution was each filled into glass bottles which were then equipped with a pump head, resulting in a gas-free chitosan-based wound spray. Similarly, the liquid-type dressing material was placed into a spraying apparatus containing a pressurized gas.

(57) The features described in the above description, claims and figures can be relevant to the invention in any combination. The reference numerals in the claims have merely been introduced to facilitate reading of the claims and are by no means meant to be limiting.