Hemostatic sponge

Abstract

The present invention provides a hemostatic porous composite sponge comprising: i) a matrix of a biomaterial; and ii) one hydrophilic polymeric component comprising reactive groups wherein i) and ii) are associated with each other so that the reactivity of the polymeric component is retained, wherein associated means that said polymeric component is coated onto a surface of said matrix of a biomaterial, or said matrix is impregnated with said polymeric material, or both.

Claims

1. A method of manufacturing a hemostatic composite, comprising: contacting a sponge comprising a matrix of a biomaterial in dried form with a reactive polymeric material in the form of a solution so that the biomaterial is impregnated with the reactive polymeric material or the reactive polymeric material is coated onto a surface of the matrix of the biomaterial, or both, wherein the reactive polymeric material comprises a polyethylene glycol (PEG) comprising two or more reactive groups selected from the group consisting of succinimidyl esters (—CON(COCH.sub.2).sub.2), aldehydes (—CHO), and isocyanates (—N═C═O), and drying the contacted biomaterial and reactive polymeric material.

2. The method of claim 1, wherein the reactive polymeric material is a reactive hydrophilic polymeric material.

3. The method of claim 1, wherein the reactive polymeric material is a single reactive hydrophilic polymeric material.

4. The method of claim 1, wherein the reactive polymeric material is a single hydrophilic polymeric material comprising electrophilic reactive groups, wherein the hydrophilic polymeric material is a hydrophilic crosslinker.

5. The method of claim 1, wherein the reactive polymeric material comprises a polyethylene glycol (PEG).

6. The method of claim 1, wherein the biomaterial is impregnated with the reactive polymeric material.

7. The method of claim 1, wherein the reactive polymeric material is coated onto a surface of the matrix of the biomaterial.

8. A hemostatic composite comprising a sponge comprising a matrix of a biomaterial that is impregnated with a reactive polymeric material obtained by the method of claim 1.

9. A hemostatic composite comprising a sponge comprising a matrix of a biomaterial that is coated with a reactive polymeric material obtained by the method of claim 1.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Hemostatic efficacy of a collagen pad coated with NHS-PEG

(2) A hemostatic pad is produced according to example 2 and coated with 14 mg/cm.sup.2 COH102 (as defined below). The hemostatic efficacy is evaluated according to the animal as described below. The bleeding is stopped 2 min after the pad application. No rebleeding is observed.

(3) FIG. 2: Hemostatic efficacy of a collagen pad impregnated with NHS-PEG

(4) A hemostatic pad is produced according to example 3 and impregnated with 8 mg/cm.sup.2 COH102. The hemostatic efficacy is evaluated according to the animal as described below. The bleeding is stopped 2 min after the pad application. No rebleeding is observed.

(5) FIG. 3: Hemostatic efficacy of a collagen pad containing oxidized cellulose fabric coated with NHS-PEG

(6) A hemostatic pad is produced according to example 5 and coated with 14 mg/cm.sup.2 COH102. The hemostatic efficacy is evaluated according to the animal as described below. The bleeding is stopped 2 min after the pad application. No rebleeding is observed.

(7) FIG. 4: Hemostatic efficacy of an oxidized cellulose fabric coated with NHS-PEG

(8) A hemostatic pad is produced according to example 6 and coated with 14 mg/cm.sup.2 COH102. The hemostatic efficacy is evaluated according to the animal as described below. The bleeding is stopped 2 min after the pad application. No rebleeding is observed.

(9) FIG. 5: Hemostatic efficacy of a collagen pad containing fucoidan as hemostasis enhancing substance coated with NHS-PEG

(10) A hemostatic pad is produced according to example 7 and coated with 14 mg/cm.sup.2 COH102. The hemostatic efficacy is evaluated according to the animal as described below. The bleeding is stopped 2 min after the pad application. No rebleeding is observed.

(11) FIG. 6: Scanning electron microscopy image (magnification: ×500) of the surface of a discontinuously coated collagen sponge

(12) FIG. 7: Scanning electron microscopy image (magnification: ×500) of the surface of a continuously coated collagen sponge

(13) FIG. 8: Gelfoam coated with 14 mg/cm.sup.2 COH102 in the liver lobe abrasion model

(14) FIG. 9: Chitoskin coated with 14 mg/cm.sup.2 COH102 in the liver lobe abrasion model The present invention is further exemplified by the following examples without being limited thereto.

(15) In the subsequent sections the following abbreviations are used: ACT activated coagulation time AcOH acetic acid NaOAc sodium acetate aq. aqueous COH102 Pentaerythritolpoly(ethyleneglycol)ether tetrasuccinimidyl glutarate=Pentaerythritol tetrakis[1-1′-oxo-5′-succinimidylpentanoate-2-poly oxoethyleneglycolejether (=an NHS-PEG with MW 10,000) EtOH ethanol PEG polyethylene glycol PET polyethylene terephthalate min minutes NHS-PEG-NHS A-[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]-ω-[6-[(2, 5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyloxy]-polyoxyethylene 8-arms-NHS-PEG Hexaglycerol octa(succinimidyloxyglutaryl)polyoxyethylene 4-arms-p-NP-PEG Pentaerythrioltetra(4-nitrophenoxycarbonyl) polyoxyethylene CHO-PEG-CHO homobifunctional aldehyd-polyethylenglycole Epoxy-PEG-Epoxy homobifunctional epoxy-polyethylenglycole 4-arm-Epoxy-PEG homomultifunctional epoxy polyethylenglycole ISC-PEG-ISC homobifunctional isocyanate-polyethylenglycole AA-dextran aldehyde-activate dextran DSS Disuccinimidyl suberate EGS Ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester)

EXAMPLES

(16) Animal Hemostasis Model for Testing the Efficacy of Hemostatic Pads of the Present Invention (Liver Surface Abrasion Model)

(17) The efficacy of the hemostatic pads of the present invention is tested in a liver surface abrasion model on heparinized (2×ACT) pigs. With a flat, round, rotating abrasion tool a circular bleeding wound (1.8 cm diameter) is created on the liver surface. A pad of the present invention (size=3×3 cm) is applied in its dry state onto the bleeding wound and hold in place by exerting slight pressure with a saline wetted gauze for 2 min. The efficacy in stopping the bleeding is evaluated.

Example 1

Preparation of Bovine Collagen Suspension

(18) 50 g of sliced bovine corium are dispersed in 500 ml of a 2M NaOH-solution and stirred approx. 90 min at 25° C. The corium is sieved out and rinsed with distilled H.sub.2O until effluent H.sub.2O reaches a pH of about 8.0. The washed corium slices are re-suspended in H.sub.2O and the pH is adjusted with HCl to approx. 2.0. The suspension obtained is stirred overnight at approx. 25° C. and a collagen solution is obtained. The solution obtained is cooled to 5° C. and the pH is adjusted with NaOH to neutral. Collagen precipitation is carried out overnight by keeping the solution at 18° C. without stirring. Precipitated collagen obtained is separated by filtration. The collagen concentration of the material obtained is determined by gravimetry. Optionally a chemical crosslinking with glutaraldehyde may be carried out in that a 1% aq. collagen suspension is prepared and 5000 ppm of glutaraldehyde are added at 12° C. The suspension obtained is stirred overnight. Crosslinked collagen obtained is filtered and washed with H.sub.2O. The collagen concentration of the material obtained is determined as described above.

Example 2

Collagen Pad Coated with NHS-PEG

(19) COH102 powder is homogeneously distributed onto one surface of a commercially available collagen sponge (Matristypt®, Dr. Suwelack Skin- and Healthcare, Germany, thickness 1 mm or 2 mm). COH102 amounts of 2 mg/cm.sup.2, 7 mg/cm.sup.2, 10 mg/cm.sup.2, 14 mg/cm.sup.2, 20 mg/cm.sup.2 are used for the coating. The COH102 powder is fixed on the surface of the sponge by melting. This is performed at 60° C. to 65° C. for 4 min by placing the sponge with the PEG powder mixture into a preheated oven.

(20) A dried sponge obtained is sealed together with a sachet of desiccant in a gas-impermeable pouch and γ-sterilized at 25 kGray.

Example 3

Collagen Pad Impregnated with NHS-PEG

(21) Aq. acidic solutions (pH 3.0, AcOH) of COH102 with concentrations of 10 mg/cm.sup.3, 20 mg/cm.sup.3, 30 mg/cm.sup.3 and 40 mg/cm.sup.3 are prepared and filled into 9×7 cm PET-trays. Commercial available bovine collagen sponges (Matristypt®), 9×7×0.1 or 0.2 cm, with the same volume as the previously filled COH102 solution are placed on the top of the solutions for impregnation for 20 min. COH102 solution is absorbed and the collagen material obtained is lyophilized. Sponges obtained can be additionally coated with COH102 as described in example 2.

(22) After lyophilization and/or coating each dried sponge obtained is sealed together with a sachet containing desiccant in a gas impermeable pouch and sterilized by γ-irradiation at 25 kGray.

Example 4

Collagen Pad Containing Oxidized Cellulose Powder Coated with NHS-PEG

(23) 0.5 g or 19 Traumastem® P powder (Bioster, Czech Republic) is homogenously distributed into 22 ml of neutral aqueous collagen suspension (2.15 mg/ml; 4.3 mg/ml and 10 mg/ml) produced according to example 1. The mixture obtained is filled into flat 9×7 cm PET trays and lyophilized. A fleece obtained has a thickness of about 3-4 mm and is coated with COH102 as described in example 2.

(24) After coating, each sponge obtained is sealed together with a sachet containing desiccant in a gas impermeable pouch and sterilized by γ-irradiation at 25 kGray.

Example 5

Collagen Pad Containing Oxidized Cellulose Fabric Coated with NHS-PEG

(25) A 6×5 cm Traumastem® TAF light-fabric (Bioster, Czech Republic) is immersed into a 1% bovine collagen suspension as described in example 1. The 6×5 cm oxidized cellulose fabric retains approximately 6 g of the collagen suspension. A fabric soaked with the collagen suspension is obtained and laid in a tray and lyophilized. A fleece obtained has a thickness of about 3-4 mm and is coated with COH102 as described in example 2.

(26) After coating, each sponge obtained is sealed together with a sachet containing desiccant in a gas impermeable pouch and sterilized by γ-irradiation at 25 kGray.

Example 6

Oxidized Cellulose Fabric Coated with NHS-PEG

(27) A double layer Traumastem® P fleece (Bioster, Czech Republic) is coated with 14 mg/cm.sup.2 COH102 as described in example 2. The thickness of the pad obtained is about 1-2 mm.

Example 7

Collagen Pad Containing Fucoidan as Hemostasis Enhancing Substance Coated with NHS-PEG

(28) A bovine collagen sponge Matristypt (9×7×0.2 cm) is impregnated with the same volume of a Fucoidan solution of A. nodosum (10 μM and 200 μM in 40 mM Ca.sup.2+-solution) and lyophilized. A sponge obtained is coated with COH102 as described in example 2.

Example 8

Collagen Pad Containing Thrombin as Hemostasis Enhancing Substance Coated with NHS-PEG

(29) A bovine collagen sponge Matristypt® (9×7×−0.2 cm) is impregnated with the same volume of a thrombin solution (500 IU/ml) and lyophilized. A sponge obtained is coated with COH102 as described in example 2.

Example 9

Sealing Efficacy of a Collagen Pad Coated with NHS-PEG

(30) A hemostatic pad coated with 14 mg/cm.sup.2 COH102 is produced according to example 2. A lesion of around 1.5 to 2 cm in diameter is set by a scalpel on the lung of a pig. A sample of 3×3 cm of the said pad is applied onto the wound and hold in place by exerting slight pressure with the aid of gauze for 2 min. The gauze is pre-wetted either with saline or basic bicarbonate solution (pH 8.3). After application, the pad is adhering firmly to the lung surface (see FIG. 6). The speed of obtaining adherence is increased using gauze wetted with bicarbonate. In order to control the air tightness and the pad-adherence to the tissue the chest is filled with Ringer's solution after 10 min. No gas leakage or detachment of the pad is observed.

Example 10

Sealing Efficacy of a Collagen Pad Impregnated with NHS-PEG

(31) A hemostatic pad impregnated with 40 mg/cm.sup.3 COH102 is produced according to example 3.

(32) A lesion of around 1.5 to 2 cm in diameter is set by a scalpel on the lung of a pig. A sample of 3×3 cm of the said pad is applied onto the wound and hold in place by exerting slight pressure with the aid of gauze for 2 min. The gauze is pre-wetted with basic bicarbonate solution (pH 8.3). After application the pad is adhering firmly to the lung surface. Air tightness and pad-adherence to the tissue are determined as described in Example 9.

Example 11

Color Marking of One Pad Surface

(33) A mask made of a stainless steel plate (1 mm thickness) with a pattern of holes is placed on one side of a 1 or 2 mm thick collagen sponge (Matristypt®, Dr. Suwelack Skin- and Healthcare, Germany). The holes of the mask have a diameter of 2 mm and are placed at a distance of 1 cm from each other in the nodes of an upright square lattice. A 0.5% aqueous Erioglaucine (Fluka, Switzerland) solution is sprayed with a standard airbrush device over the holes of the mask. The mask is removed and a collagen sheet with the blue dot pattern obtained is dried at ambient atmosphere, in a vacuum oven or in a desiccator. The dot pattern on one side has the role to distinguish the active and inactive surface of a coated pad. It is possible to apply the coating either on the dotted side or the non-dotted side.

Example 12

Preparation of a Fibrin Fleece

(34) A solution of 2.5 mg/ml of fibrinogen, 10 mM Tris HCl, 150 mM NaCl, pH 7.4 and an equal volume of 55 IU thrombin/ml, 10 mM CaCl.sub.2 are mixed using a static mixer and immediately filled into a tray at a height of 0.7 cm. A fibrin clot is obtained in the tray. By freeze-drying of the clot a fibrin fleece is obtained.

Example 13

Preparation of Collagen Pad Coated with NHS-PEG-NHS and its Testing in Animal Model

(35) On the non-colored side of a 6×6 cm collagen pad (made as described in example 11) 14 mg/cm.sup.2 and 28 mg/cm.sup.2 of bifunctional NHS-PEG-NHS (MW 10000, NOF Corporation, Japan) are homogeneously distributed and fixed by melting. This is performed at approx. 70° C. for 4 min by placing the sponge coated with the PEG powder into a preheated oven. Sponges obtained are sealed together with a sachet containing desiccant in a gas-impermeable pouch.

(36) The hemostatic performance of said pads are tested in pig in the liver abrasion model as described above. After 2 minutes hemostasis is achieved. No rebleeding after 10 minutes is observed. The adherence of the pad on the tissue is sufficient.

Example 14

Preparation of Collagen Pad Coated with 8-arm-NHS-PEG and its Testing in Animal Model

(37) On the non-colored side of a 6×6 cm collagen pad, made as described in example 11, 14 mg/cm.sup.2 8-arm-NHS-PEG (MW 15000, NOF Corporation, Japan) are homogeneously distributed and fixed by melting. This is performed at 65° C. for 4 min by placing the sponge with the PEG powder into a preheated oven.

(38) A sponge obtained is sealed together with a sachet containing desiccant in a gas-impermeable pouch.

(39) The hemostatic performance of said pad is tested in pig in the liver abrasion model as described above. After 2 minutes hemostasis is achieved. No rebleeding after 10 minutes is observed. The adherence of the pad on the tissue is sufficient.

Example 15a

Preparation of Collagen Pad Coated with 4-arm-p-NP-PEG and its Testing in Animal Model

(40) On the non-colored side of a 6×6 cm collagen pad, made as described in example 11, 14 mg/cm.sup.2 4-arm-p-NP-PEG (MW 10000, NOF Corporation, Japan) are homogeneously distributed and fixed by melting. This is performed at 65° C. for 4 min by placing the sponge with the PEG powder into a preheated oven.

(41) A sponge obtained is sealed together with a sachet containing desiccant in a gas-impermeable pouch.

(42) The hemostatic performance of the said pad is tested in pig in the liver abrasion model as described above. After 2 minutes hemostasis is achieved. No rebleeding after 10 minutes is observed. The adherence of the pad on the tissue is not sufficient.

Example 15b

Preparation of Collagen Pad Coated with 4-arm-p-NP-PEG and its Testing in Animal Model

(43) The hemostatic performance of the pad as prepared in Ex. 15a is tested in pig in the liver abrasion model as described above but with the modification, that the pad is applied with gauze pre-wetted with basic 8% Na-bicarbonate solution. After 2 minutes hemostasis is achieved. No rebleeding after 10 minutes is observed. The adherence of the pad on the tissue is sufficient.

Example 16a

Preparation of Collagen Pad Coated with CHO-PEG-CHO and its Testing in Animal Model

(44) On the non-colored side of a 6×6 cm collagen pad, made as described in example 11, 9.5 mg/cm.sup.2 CHO-PEG-CHO (MW 3400, Interchim, France) are homogeneously distributed and fixed by melting. This is performed at 70° C. for 4 min by placing the sponge with the PEG powder into a preheated oven.

(45) A sponge obtained is sealed together with a sachet containing desiccant in a gas-impermeable pouch.

(46) The hemostatic performance of said pad is tested in pig in the liver abrasion model as described above. After 2 minutes hemostasis is achieved. No rebleeding after 10 minutes is observed. The adherence of the pad on the tissue is sufficient.

Example 16b

Preparation of Collagen Pad Coated with CHO-PEG-CHO and its Testing in Animal Model

(47) The hemostatic performance of the pad as prepared in Ex. 16a is tested in pig in the liver abrasion model as described above but with the modification, that the pad is applied with gauze pre-wetted with basic Na-bicarbonate solution. After 2 min hemostasis is achieved. No rebleeding after 10 min is observed. The adherence of the pad on the tissue is sufficient.

Example 17a

Preparation of Collagen Pad Coated with Epoxy-PEG-Epoxy and its Testing in Animal Model

(48) On the non-colored side of a 6×6 cm collagen pad made, as described in example 11, 9.5 mg/cm.sup.2 Epoxy-PEG-Epoxy (MW 3400, Interchim, France) are homogeneously distributed and fixed by melting. This is performed at 70° C. for 4 min by placing the sponge with the PEG powder into a preheated oven.

(49) A sponge obtained is sealed together with a sachet containing desiccant in a gas-impermeable pouch.

(50) The hemostatic performance of said pad is tested in pig in the liver abrasion model as described above. After 2 min no hemostasis is achieved. The adherence of the pad on the tissue is not sufficient.

Example 17b

Preparation of Collagen Pad Coated with Epoxy-PEG-Epoxy and its Testing in Animal Model

(51) The hemostatic performance of the pad as prepared in Ex. 17a is tested in pig in the liver abrasion model as described above but with the modification, that the pad is applied with gauze pre-wetted with basic Na-bicarbonate solution. After 2 min hemostasis is achieved. No rebleeding after 5 min is observed. The adherence of the pad on the tissue is sufficient.

Example 18

Preparation of Collagen Pad Coated with 4-arm-Epoxy-PEG and its Testing in Animal Model

(52) On the non-colored side of a 6×6 cm collagen pad, made as described in example 11, 14 mg/cm.sup.2 4-arm-epoxy-PEG (MW 10000, Interchim, France) are homogeneously distributed and fixed by melting. This is performed at 70° C. for 4 min by placing the sponge with the PEG powder into a preheated oven.

(53) A sponge obtained is sealed together with a sachet containing desiccant in a gas-impermeable pouch.

(54) The hemostatic performance of said pad is tested in pig in the liver abrasion model as described above, but with the modification, that the pad is applied with gauze pre-wetted with basic Na-bicarbonate solution. After 2 min hemostasis is achieved. No rebleeding after 5 min is observed.

(55) The adherence of the pad on the tissue is sufficient.

Example 19

Preparation of Collagen Pad Coated with ISC-PEG-ISC and its Testing in Animal Model

(56) On the non-colored side of a 6×6 cm collagen pad, made as described in example 11, 9.5 mg/cm.sup.2 ISC-PEG-ISC (MW 3400, Interchim, France) are homogeneously distributed and fixed by melting. This is performed at 70° C. for 4 min by placing the sponge with the PEG powder into a preheated oven.

(57) A sponge obtained is sealed together with a sachet containing desiccant in a gas-impermeable pouch.

(58) The hemostatic performance of said pad is tested in pig in the liver abrasion model as described above. After 2 min hemostasis is achieved. No rebleeding after 10 min is observed. The adherence of the pad on the tissue is sufficient.

Example 20

Preparation of Collagen Pad Coated with AA-dextran and its Testing in Animal Model

(59) On the non-colored side of a 6×6 cm collagen pad made as described in example 11 14 mg/cm.sup.2 of a mixture of 0.1 mg/cm.sup.2 AA-dextran (MW 40000, Pierce, USA) and 13.9 mg/cm.sup.2 unsubstituted PEG (MW 10000, Sigma Aldrich, Germany) are homogeneously distributed and fixed by melting. This is performed at 80° C. for 4 min by placing the sponge with the powder mixture into a preheated oven.

(60) A sponge obtained is sealed together with a sachet containing desiccant in a gas-impermeable pouch.

(61) The hemostatic performance of said pad is tested in pig in the abrasive liver lobe model as described above but with the modification, that the pad is applied with gauze pre-wetted with basic Na-bicarbonate solution. After 2 min hemostasis is achieved. No rebleeding after 10 min is observed. The adherence of the pad on the tissue is sufficient.

Example 21a

Preparation of Collagen Pad Coated with DSS and its Testing in Animal Model

(62) On the non-colored side of a 6×6 cm collagen pad made as described in example 11 20 mg/cm.sup.2 of a 1:1 mixture of DSS (MW 368.35, Sigma Aldrich, Germany) and unsubstituted PEG (MW 10000, Sigma Aldrich, Germany) are homogeneously distributed and fixed by melting. This is performed at 80° C. for 4 min by placing the sponge with the powder mixture into a preheated oven.

(63) A sponge obtained is sealed together with a sachet containing desiccant in a gas-impermeable pouch.

(64) The hemostatic performance of said pad is tested in pig in the abrasive liver lobe model as described above. After 2 min hemostasis is not achieved. The adherence of the pad on the tissue is not sufficient.

Example 21b

Preparation of Collagen Pad Coated with DSS and its Testing in Animal Model

(65) The hemostatic performance of the pad as prepared in Ex. 21 a is tested in pig in the abrasive liver lobe model as described above but with the modification, that the pad is applied with gauze pre-wetted with basic bicarbonate solution. After 2 min hemostasis is achieved. No rebleeding after 10 min is observed. The adherence of the pad on the tissue is sufficient.

Example 22a

Preparation of Collagen Pad Coated with EGS and its Testing in Animal Model

(66) On the non-colored side of a 6×6 cm collagen pad made as described in example 11 26 mg/cm.sup.2 of a 1:1 mixture of EGS (MW 456.36, Sigma Aldrich, Germany) and unsubstituted PEG (MW 10000, Sigma Aldrich, Germany) are homogeneously distributed and fixed by melting. This is performed at 80° C. for 4 min by placing the sponge with the powder mixture into a preheated oven.

(67) A sponge obtained is sealed together with a sachet containing desiccant in a gas-impermeable pouch.

(68) The hemostatic performance of said pad is tested in pig in the liver abrasion model as described above. After 2 min hemostasis is not achieved. The adherence of the pad on the tissue is not sufficient.

Example 22b

Preparation of Collagen Pad Coated with EGS and its Testing in Animal Model

(69) The hemostatic performance of the pad as prepared in Ex. 22a is tested in pig in the liver abrasion model as described above but with the modification, that the pad is applied with gauze pre-wetted with basic Na-bicarbonate solution. After 2 min hemostasis is achieved. No rebleeding after 10 min is observed. The adherence of the pad on the tissue is sufficient.

Example 23

Fibrin Fleece Coated with NHS-PEG

(70) On one side of the fibrin fleece, made as described in example 12, 14 mg/cm.sup.2 of COH102 are homogeneously distributed and fixed by melting. This is performed at 65° C. for 4 min by placing the sponge with the PEG powder into a preheated oven.

(71) A sponge obtained is sealed together with a sachet containing desiccant in a gas-impermeable pouch.

(72) The hemostatic performance of said pad is tested in pig in the liver abrasion model as described above. After 2 min hemostasis is achieved. No rebleeding after 10 min is observed. The adherence of the pad on the tissue is sufficient.

Example 24

Correlation Between the Adherence Force to the Tissue and the Cross-Linker Used for Collagen Pad Coating

(73) After application of the pads to the bleeding tissue in the liver abrasion model the adherence of the pad to the liver tissue is evaluated. Slight tangential force is applied with the lateral part of a forceps. Presence of adherence (bonding to the tissue) is considered if it is not possible to displace the pad from the site of application. Adherence score: 1=no displacement at 5 min after application; 2=no displacement 10 min after application; 3=displacement (no adherence) 10 min after application.

(74) TABLE-US-00001 Example No. Cross-linker Adherence score 13 NHS-PEG-NHS 1 14 8-arms-NHS-PEG 1 15a 4-arms-p-NP-PEG 3 15b 4-arms-p-NP-PEG - basic application 2 16a CHO-PEG-CHO 1 16b CHO-PEG-CHO - basic application 2 17a Epoxy-PEG-Epoxy 3 17b Epoxy-PEG-Epoxy - basic application 2 18 4-arm-Epoxy-PEG - basic application 2 19 ISC-PEG-ISC 1 20 AA-dextran - basic application 1 21a DSS 3 21b DSS - basic application 2 22a EGS 3 22b EGS - basic application 2

Example 25

Chitosan/Gelatine Sponge Coated with NHS-PEG and its Testing in Animal Model

(75) On a commercially available chitosan/gelatin sponge (Chitoskin®, Beese Medical, Germany) 14 mg/cm.sup.2 of COH102 are homogeneously distributed and fixed by melting. This is performed at 65° C. for 4 min by placing the sponge with the PEG powder into a preheated oven.

(76) A sponge obtained is sealed together with a sachet containing desiccant in a gas-impermeable pouch.

(77) The hemostatic performance of said pad is tested in pig in the liver abrasion model as described above. After 2 min hemostasis is achieved. No rebleeding after 10 min is observed (FIG. 9).

(78) The adherence of the pad on the tissue is sufficient.

Example 26

Preparation of a Gelatin Pad Coated with NHS-PEG and its Testing in Animal Model

(79) On a commercially available gelatin sponge (Gelfoam®, Pfizer, USA) 14 mg/cm.sup.2 COH102 are homogeneously distributed and fixed by melting. This is performed at approx. 70° C. for 4 min by placing the sponges coated with the PEG powder into a preheated oven.

(80) The sponge obtained is sealed together with a sachet containing desiccant in a gas-impermeable pouch.

(81) The hemostatic performance of said pads are tested in pig in the liver surface abrasion model as described above. After 10 minutes hemostasis is not achieved due to a lack of adherence on the tissue and slow liquid uptake of the sponge.

Example 27

Water Uptake Velocities

(82) A 2×2 cm piece of a dry collagen sponge (Matristypt®, Dr. Suwelack, Germany) or of a dry cross-linked gelatin sponge (Gelfoam®, Pfizer) are placed onto the surface of distilled H.sub.2O into a beaker. The dry sponges are floating on the water surface and take up water over the 2×2 cm contact surface. After 6 s Matristypt® is totally soaked by H.sub.2O and removed from the water surface. The thicker Gelfoam® sponge is not totally soaked by H.sub.2O after 13 s, but removed after 13 s from the water surface. From the weights of the 2×2 cm sponges before and after the contact with the water surface, the time of contact with the water surface and the area of contact with the water surface the initial water uptake velocities of the sponges (in mg water/s) per surface of contact (in cm.sup.2) is calculated. The initial water uptake velocities are 35 mg×cm.sup.−1s.sup.−1 for Matristypt® and 0.8 mg×cm.sup.−1s.sup.−1 for Gelfoam®.