HK1193774B - Hemostatic textile - Google Patents

Description

Hemostatic fabric

This application is a divisional application of the Chinese invention patent application No. 200780006941.4.

Background of the Invention

1. Field of the Invention

The present invention relates to fabrics such as bandages, sutures or fabrics and, more particularly, to hemostatic fabrics that include an agent that rapidly controls bleeding and that can be stored for extended periods of time.

2. Description of Related Technology

Although great progress has been made in understanding the pathophysiological process relevant to surface (local) hemostasis, it is still very necessary to have a material that can be used for hemostasis of bleeding sites. Traumatic injury is the leading cause of death of individuals under 44 years old (Bozeman, W.Shock, Hemorrhage (2001)). In the U.S., about half of the 100,000 deaths from traumatic injury are caused each year, or 50,000 cases are due to hemorrhage (Peng, R., Chang, C., Gilmore, D. & Bongard, F.Am Surg Vol. 64 950-4 (1998)), and about the same number of hemorrhage patients survive after massive red blood cell transfusion (Vaslef, S., Knudsen, N., Neligan, P., and Sebastian, M.J.Trauma-Inj. Inf. Crit. Care Vol. 53 291-296 (2002)). Therefore, about 100,000 patients are in urgent need of bleeding control each year in the U.S. The situation is equally critical in combat casualty care; in a recent summary of military casualties (Burlingame, B. DOD's experiences in Afghanistan Advanced Technological Applications for Combat Casualty Care 2002 Conference in www.usaccc.org (2002)), control of non-compressible bleeding was identified as the single most important need in military emergency medicine. The standard of care is often to use a tourniquet to control "compressible bleeding" and then gauze to control the remaining "non-compressible bleeding." However, persistent blood loss through the gauze is a major cause of mortality and morbidity.

The prior art is full of patients using various forms of bandages. For example, U.S. Pat. No. 3,419,006 to King discloses a sterile transparent wound dressing made of a hydrophilic polymer gel of an insoluble polymer, and U.S. Pat. No. 4,323,061 to Usukura discloses a rigid bandage made of glass fibers and non-glass fibers. Furthermore, various methods have been attempted to quickly stop bleeding in injured individuals. Some of these methods include articles, such as bandages, supplemented with substances that chemically accelerate the body's natural clotting process. Examples of these articles include:

U.S. 3,328,259 to Anderson discloses a bandage or wound dressing incorporating polymers such as sodium carboxymethylcellulose, hydroxyethylcellulose, polyethylene oxide, polyvinylpyrrolidone, and the like.

U.S. 4,192,299 to Sabatano discloses a bandage including a packet containing an antimicrobial substance.

Sawyer, U.S. 4,390,519, discloses a bandage in the form of a sponge comprising collagen or a collagen-like substance.

U.S. Patent No. 4,453,939 to Zimmerman et al. discloses a composition for use as a wound dressing consisting of collagen, fibrinogen, and thrombin.

U.S. Patent No. 4,606,337 to Zimmerman et al. discloses a resorbable sheet for sealing and healing wounds composed of a glycoprotein matrix containing fibrinogen and thrombin.

U.S. 4,616,644 to Saferstein et al. discloses an adhesive bandage comprising high molecular weight polyethylene oxide as a hemostatic agent.

Bell et al., U.S. 5,800,372, discloses a dressing made of absorbent polymers, including microfibrillar collagen.

U.S. Pat. No. 5,902,608 to Read et al. discloses a surgical aid, such as a bandage, gauze, suture, etc., comprising fixed dried blood cells expressing platelet-derived growth factor.

U.S. 6,638,296 to Levinson discloses a bandage including a pad containing glucosamine or a glucosamine derivative.

MacPhee et al., U.S. 6,762,336 and International Patent Application Publication WO/99/59647, disclose a multilayer bandage comprising a thrombin layer sandwiched between two fibrinogen layers.

U.S. 6,897,348 to Malik discloses an adhesive bandage comprising an antimicrobial agent and a hemostatic agent (e.g., chitosan, niacinamide, or ascorbic acid), or a single wound healing agent comprising an antimicrobial and hemostatic additive (e.g., chitosan, niacinamide, or ascorbate).

U.S. Pat. No. 6,891,077 to Rothwell et al. discloses a fibrinogen bandage comprising a procoagulant such as propyl gallate, gallic acid or a derivative thereof. Other ingredients such as thrombin or an antimicrobial agent may also be included.

International Patent Application Publication No. WO 97/28823 to New Generation Medical Corporation discloses a hemostatic bandage comprising powdered fibrinogen and thrombin adhered to a viscous or non-viscous adhesive such as a viscous polysaccharide, glycol or petroleum jelly.

Despite great progress in understanding the pathophysiological processes associated with hemostasis, tissue remodeling, and breakdown at the site of injury, there remains a significant need for substances that can be applied to the site of injury to promote these processes. The present invention is believed to be an answer to this need.

Summary of the Invention

In one aspect, the present invention relates to a hemostatic fabric comprising: a material comprising a combination of glass fibers and one or more secondary fibers, wherein the secondary fibers are selected from silk fibers; ceramic fibers; virgin or regenerated bamboo fibers; cotton fibers; rayon fibers; linen fibers; ramie fibers; jute fibers; sisal fibers; flax fibers; soy fibers; corn fibers; hemp fibers; lyocel fibers; wool; lactide and/or glycolide polymers; lactide/glycolide copolymers; silicate fibers; polyamide fibers; feldspar fibers; zeolite fibers, fibers containing zeolites, acetate fibers; and combinations thereof; when applied to a wound, the hemostatic fabric is capable of activating the hemostatic system in the body.

In another aspect, the present invention relates to a hemostatic fabric comprising: a material comprising a combination of about 65% by weight glass fibers and about 35% by weight virgin or regenerated bamboo fibers; the hemostatic fabric is capable of activating the body's hemostatic system when applied to a wound.

In another aspect, the present invention relates to a hemostatic fabric comprising: a material comprising a combination of glass fibers and one or more secondary fibers, wherein the secondary fibers are selected from silk fibers; polyester fibers; nylon fibers; ceramic fibers; virgin or regenerated bamboo fibers; cotton fibers; rayon fibers; linen fibers; ramie fibers; jute fibers; sisal fibers; flax fibers; soy fibers; corn fibers; hemp fibers; lyocel fibers; wool; lactide and/or glycolide polymers; lactide/glycolide copolymers; silicate fibers; polyamide fibers; feldspar fibers; zeolite fibers, fibers containing zeolites; acetate fibers; and combinations thereof; and thrombin or a portion containing thrombin; when applied to a wound, the hemostatic fabric is capable of activating the hemostatic system in the body.

In another aspect, the present invention relates to a hemostatic fabric comprising: a material comprising a combination of about 65% by weight glass fibers and about 35% by weight virgin or regenerated bamboo fibers, based on the total weight of the fabric; and about 0.1 to about 5% by weight thrombin or a portion comprising thrombin; when applied to a wound, the hemostatic fabric is capable of activating the hemostatic system in the body.

In another aspect, the present invention relates to a hemostatic fabric comprising: a material comprising a combination of glass fibers and one or more secondary fibers, wherein the secondary fibers are selected from silk fibers; polyester fibers; nylon fibers; ceramic fibers; virgin or regenerated bamboo fibers; cotton fibers; rayon fibers; linen fibers; ramie fibers; jute fibers; sisal fibers; flax fibers; soy fibers; corn fibers; hemp fibers; lyocel fibers; wool; lactide and/or glycolide polymers; lactide/glycolide copolymers; silicate fibers; polyamide fibers; feldspar fibers; zeolite fibers, fibers containing zeolites; acetate fibers; and combinations thereof; and one or more hemostatic agents selected from RL platelets, RL blood cells; fibrin, and fibrinogen; when applied to a wound, the hemostatic fabric is capable of activating the hemostatic system in the body.

In another aspect, the present invention relates to a hemostatic fabric comprising: a material comprising a combination of about 65% by weight glass fiber and about 35% by weight virgin or regenerated bamboo fiber; and one or more hemostatic agents selected from RL platelets, RL blood cells, fibrin, and fibrinogen, wherein the RL platelets and RL blood cells comprise about 0.1 to about 20 wt %, and the fibrin and fibrinogen comprise about 0.1 to about 5 wt %, based on the total weight of the fabric; when applied to a wound, the hemostatic fabric is capable of activating the hemostatic system in the body.

In another aspect, the present invention relates to a hemostatic fabric comprising: a material comprising a combination of glass fibers and one or more secondary fibers, wherein the secondary fibers are selected from silk fibers; polyester fibers; nylon fibers; ceramic fibers; virgin or regenerated bamboo fibers; cotton fibers; rayon fibers; linen fibers; ramie fibers; jute fibers; sisal fibers; flax fibers; soy fibers; corn fibers; hemp fibers; lyocel fibers; wool; lactide and/or glycolide polymers; lactide/glycolide copolymers; silicate fibers; polyamide fibers; feldspar fibers; zeolite fibers, fibers containing zeolites; acetate fibers; and combinations thereof; and thrombin or a portion containing thrombin; and one or more hemostatic agents selected from RL platelets, RL blood cells; fibrin and fibrinogen; when applied to a wound, the hemostatic fabric is capable of activating the hemostatic system in the body.

In another aspect, the present invention relates to a hemostatic fabric comprising: a material comprising a combination of about 65% by weight glass fiber and about 35% by weight virgin or regenerated bamboo fiber; about 0.1 to about 5% by weight of thrombin or a portion comprising thrombin, based on the total weight of the fabric; and one or more hemostatic agents selected from RL platelets, RL blood cells, fibrin and fibrinogen, wherein the RL platelets and RL blood cells account for about 0.1 to about 20 wt % and the fibrin and fibrinogen account for about 0.1 to about 5 wt % based on the total weight of the fabric; when applied to a wound, the hemostatic fabric is capable of activating the hemostatic system in the body.

In another aspect, the present invention relates to a method for preparing a hemostatic fabric, comprising the steps of: (1) contacting a material comprising a combination of glass fibers and one or more secondary fibers, wherein the secondary fibers are selected from silk fibers; polyester fibers; nylon fibers; ceramic fibers; virgin or regenerated bamboo fibers; cotton fibers; rayon fibers; linen fibers; ramie fibers; jute fibers; sisal fibers; flax fibers; soy fibers; corn fibers; hemp fibers; lyocel fibers; wool; lactide and/or glycolide polymers; lactide/glycolide copolymers; silicate fibers; polyamide fibers; feldspar fibers; zeolite fibers, fibers containing zeolites; acetate fibers; and combinations thereof, with thrombin or a portion containing thrombin and an optional hemostatic agent selected from RL platelets, RL blood cells; fibrin, fibrinogen, and combinations thereof to form a wet matrix; and (2) drying the wet matrix to form a hemostatic fabric.

In another embodiment, the present invention relates to a method for preparing a hemostatic fabric, comprising the following steps: (1) contacting a material comprising a combination of glass fibers and one or more secondary fibers, wherein the secondary fibers are selected from silk fibers; polyester fibers; nylon fibers; ceramic fibers; virgin or regenerated bamboo fibers; cotton fibers; rayon fibers; linen fibers; ramie fibers; jute fibers; sisal fibers; flax fibers; soy fibers; corn fibers; hemp fibers; lyocel fibers; wool; lactide and/or glycolide polymers; lactide/glycolide copolymers; silicate fibers; polyamide fibers; feldspar fibers; zeolite fibers, fibers containing zeolites; acetate fibers; and combinations thereof, with platelet-rich plasma containing platelets; (2) crosslinking the platelets; and (3) optionally contacting thrombin or a portion containing thrombin with the fabric to form the hemostatic fabric.

These and other aspects will become apparent upon reading the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood when taken in conjunction with the following drawings, in which:

FIG1 shows a representative thrombin generation curve when using one embodiment of the present invention;

FIG2 shows the time for thrombin generation when using an embodiment of the present invention;

FIG3 shows the thromboelastography analysis of the material according to the present invention;

Figure 4 shows a comparison of blood cells on double fiber and gauze;

FIG5 shows the interaction between red blood cells (RBCs) and a substance according to the present invention;

Figure 6 shows platelet activation on glass fibers used in the present invention; and

FIG7 shows the total blood loss using the material of the present invention.

Detailed Description of the Invention

The inventors of the present invention unexpectedly discovered that a hemostatic fabric can be prepared from a composite of glass fiber and one or more accessory fibers. The hemostatic fabric made from the composite of fibers shows excellent hemostatic properties and liquid absorption capacity. In order to further enhance the hemostatic properties of the hemostatic fabric made from the composite, other blood factors can be added, such as thrombin, lyophilized blood cells, lyophilized platelets, fibrin, fibrinogen, or a combination thereof. These other factors assist in activating the body's natural hemostatic cascade, resulting in substances that can quickly stop bleeding. The inventors have discovered that the combination of glass fiber, accessory fibers, and other blood factors has produced a new, fast-stopping hemostatic fabric that can be used in situations where there is heavy bleeding or when the patient cannot be immediately taken to a hospital or trauma center.

The hemostatic fabric of the present invention offers significant advantages over existing products for activating hemostasis. The present invention is capable of rapidly activating the body's natural hemostatic system, such as the coagulation cascade, by providing a localized high concentration of substances that activate the cascade. Furthermore, by utilizing lyophilized blood proteins, the hemostatic fabric of the present invention can be stored for extended periods of time in a dry state, ready for immediate use. This aspect is particularly advantageous because existing products and systems require hydrated proteins for activation.

As described above, one embodiment of the present invention is a hemostatic fabric matrix comprising a material comprising a combination of glass fibers and one or more secondary fibers. Each of these components will be discussed in more detail below.

The glass fiber component is preferably a fiber glass prepared by extrusion or electrospinning, with a fiber diameter of 5 nanometers to 15 microns. Types of glass that can be considered for use in the present invention include, but are not limited to, borosilicate glass with low sodium oxide content, borosilicate glass, lead glass, aluminosilicate, alkali barium silicate, transparent quartz, chalcogenide glass, phosphate glass, and bioactive glass sold under the trade name "BIOGLASS". The specifications of the glass fiber component can be described by conventional nomenclature, including the following designations: B (3.5 micron diameter); C (4.5 micron diameter); D (5 micron diameter); DE (6 micron diameter); E (7 micron diameter); G (9 micron diameter); H (10 micron diameter); or K (13 micron diameter). In addition, the thread count of the glass fiber component can range from 900 to 37. The glass fiber can be of any grade, electrical ("E"), chemical ("C"), or high-strength ("S"), and the filaments can be arranged in any pattern, such as continuous, U-shaped, or textured. The glass fibers can be used singly or in strands of 2 to 20 or more fibers. Fiberglass material can be purchased from various suppliers, such as Owens Corning, and can be commercially available as grade G75, E-grade fiberglass, and the like, as designated above.

The secondary fibers used in the fabrics of the present invention generally include any other fibers that, in combination with the glass fibers, impart absorbency, softness, and additional hemostatic activity to the fabric. As described in detail below, the use of secondary fibers with excellent absorbency also facilitates the incorporation of additional hemostatic factors into the fabric. Examples of useful secondary fibers include, but are not limited to, silk fibers; polyester fibers; nylon fibers; ceramic fibers; polysaccharide fibers, including plant fibers such as virgin or regenerated (e.g., chemically treated) bamboo, cotton, rayon, linen, ramie, jute, sisal, flax, soy, corn, hemp, and lyocel; animal fibers such as wool; lactide and/or glycolide polymers; lactide/glycolide copolymers; silicate fibers; polyamide fibers; feldspar fibers; zeolite fibers, fibers containing zeolites; acetate fibers; and plant fibers that have been genetically engineered to express mammalian coagulation proteins or mammalian vascular factors. Other secondary fibers that are suitable for use in the present invention are fibers covalently modified with polymers (e.g., polyvinyl alcohol) that promote water absorption and polymers that comprise the molecular moiety (e.g., linear or cyclic-arginine-glycine-aspartic acid-part that are for example found in eptifibatide) that activates the hemostatic system. Preferred secondary fibers include plant fibers such as primary or regenerated (e.g., chemically treated) bamboo fiber, cotton fiber, etc., which have very high hygroscopicity and can activate the endogenous coagulation cascade. Conventional methods can be used to include that ring, open (OE) rotor or air jet rotation prepare secondary fibers, and the counting range can be 1/1 to 100/1Ne.

Those skilled in the art will appreciate that the secondary fibers can be used alone or in a combination of two, three, four or more in a mixed or stranded state. In addition, any type of secondary fiber combination can be used. For example, in one embodiment, two or more secondary fibers can be prepared separately and then mixed or stranded together to form a composite yarn. In another embodiment, the secondary fibers can form a conjugate comprising blocks of selected types of fibers, for example, alternating blocks of polyester and polysaccharide. In another embodiment, the secondary fibers can form a uniform combination of different lines.

Based on the gross weight of dry fabric, the relative amount of glass fiber and secondary fiber can be wide range, for example, about 0.1 to 99.9wt% glass fiber and about 99.9% to 0.1% weight secondary fiber. The preferred amount range of these materials is about 30 to 80wt% glass fiber and about 70 to 20wt% secondary fiber, more preferably about 50 to 80wt% glass fiber and about 50 to 20wt% secondary fiber. The example of the useful ratio of glass and secondary fiber in hemostatic fabric of the present invention comprises about 50wt% glass fiber and about 50wt% secondary fiber; about 40wt% glass fiber and about 60wt% secondary fiber; about 30wt% glass fiber and about 70wt% secondary fiber; or about 20wt% glass fiber and about 80wt% secondary fiber. A kind of particularly useful combination is about 65% weight glass fiber and 35% weight bamboo fiber. The glass fiber component and secondary fiber component can be combined by conventional methods such as weaving, braiding or knitting methods, or can be used with non-knitted state.

In use, the hemostatic fabric of the present invention can take any configuration. In one embodiment, the hemostatic fabric is composed of a hemostatic layer designed to promote hemostasis and an outer layer that is responsible for surface structuring, moisture transfer, liquid absorption, and bacterial protection. In another embodiment, the hemostatic fabric is composed of three layers: a hemostatic layer designed to promote hemostasis, a middle layer for providing bandage strength and elasticity, and an outer layer that is responsible for surface structuring, moisture transfer, liquid absorption, and bacterial protection. Other configurations can be envisioned by those skilled in the art.

The hemostatic fabric of the present invention may be treated with various agents that enhance its effectiveness. Examples of other agents include organic or inorganic compounds that are antimicrobial or antimicrobial; organic or inorganic compounds that covalently react with blood coagulation proteins; organic or inorganic compounds that covalently react with injured tissue to form covalent bonds to enhance tissue adhesion; organic or inorganic compounds that polymerize to form a three-dimensional polymer network at or above the wound; imaging agents such as ultrasound contrast agents (e.g., gas-filled microbubbles, metal nanoparticles, etc.), radiation shielding agents (e.g., iodinated small molecules such as iopromide, iodinated high molecular weight polymers, etc.), and magnetic resonance probes (e.g., Feride nanoparticles, superparamagnetic metal nanoparticles, diethylenetriaminepentaacetic acid (DTPA)-chelated gadolinium and polymers containing DTPA-chelated gadolinium, etc.).

Other agents that may be included in the hemostatic fabric of the present invention include skin conditioners such as aloe vera, vitamin E, coenzyme Q, collagen, etc.; anti-inflammatory agents such as aspirin, ibuprofen, acetaminophen, vitamin C, COX-2 inhibitors, steroids, etc.; analgesics such as lidocaine, tetracaine, opioids, cocaine, antihistamines, etc.; antibacterial or antifungal agents such as bacitracin, silver salts, iodides, etc.; vasoconstrictors such as epinephrine, norepinephrine, vasopressin, hemoglobin, endothelin, thromboxane, NO scavengers, etc.; growth factors such as MMP inhibitors, PDGF, etc.; anti-scar agents such as IL-11, anti-keloid agents, etc.; burning agents that undergo an exothermic reaction upon rehydration, such as zeolites; water-absorbing dehydrating agents such as dextran; anti-thrombotic agents, such as zeolites, dextran sulfate, polyphosphates, mineral interfaces, phosphatidylserine, calcium, etc.

The fabric substrate of the present invention can also include other factors that activate the natural hemostatic system in vivo and contribute to rapid hemostasis thus. These other factors include thrombin or the plasma fraction that includes thrombin, the platelets of the lyophilized (RL) of rehydration, RL blood cells, fibrin, fibrinogen and their combination. In a preferred embodiment, thrombin is incorporated into the fabric to give it additional hemostatic activity. Thrombin can be (natural separation, reorganization etc.) in any source or can be the form of plasma fraction or serum, wherein plasma fraction or serum comprise thrombin and other coagulation factors such as factor XII, factor XIIa, factor XI, factor XIa, factor XIII, factor XIIIa, factor IX, factor IXa, factor VIII, factor VIIIa, factor vWF, factor V, factor Va, factor X, factor Xa and combination thereof or other coagulation cofactors such as components of animal venom, for example retardin, or vasoactive agent such as endothelin, thromboxane, nitrous oxide (NO) scavenger or its combination. When incorporated into the fabrics of the present invention, these agents or any of the agents listed above may be in dry or liquid form.

It is contemplated that the thrombin used in the fabric of the present invention can be in any form, including highly purified thrombin IIa from humans or animals, genetically modified plants or other natural or recombinant protein expression systems. In addition, partially purified thrombin from humans or animals, genetically modified plants or other natural or recombinant protein expression systems can be used in the present invention. It is contemplated that the thrombin used in the present invention can also be contained in purified or partially purified serum or plasma. In one embodiment, the thrombin used in the fabric of the present invention is a partially purified serum portion comprising thrombin IIa.

The preferred amount of thrombin in the fabric of the present invention ranges from about 0.01 wt% to about 10 wt% based on the total weight of the dry fabric. The more preferred amount of thrombin included in the fabric of the present invention ranges from about 0.05 wt% to about 7 wt%, and most preferably from about 0.1 wt% to about 5 wt%, based on the total weight of the dry fabric.

As explained in more detail in the examples hereinafter, in order to prepare a hemostatic fabric that comprises thrombin, the fabric substrate is immersed in a solution that comprises thrombin, frozen and lyophilized. In the soaking solution, a preservative such as glycerol, propylene glycol, polyethylene glycol (PEG), trehalose or the like can be included to prevent the fabric from becoming brittle or becoming similar to chalk during lyophilization. Generally, the concentration range of the preservative in the thrombin solution is approximately 20% (v/v) maximum. In a preferred embodiment, approximately 12% (v/v) of glycerol is used.

In another preferred embodiment, one or more rehydrated lyophilized (RL) platelets, RL blood cells, fibrin or fibrinogen are incorporated into the fabric to impart additional hemostatic activity. Rehydrated lyophilized blood cells and rehydrated platelets and methods for their preparation are known in the art. See, for example, U.S. 4,287,087; 5,651,966; 5,891,393; 5,902,608; 5,993,804; all of which are incorporated herein by reference. Briefly, RL platelets are prepared by isolating the platelets, exposing them to a fixative such as formaldehyde, and drying them. RL platelets can also be purchased commercially from Entegrion, Inc. (Research Triangle Park, NC) under the trade name "STASIX." Methods for isolating and purifying fibrin and fibrinogen are also known in the art.

Briefly, to prepare RL blood cells, blood can be obtained from healthy volunteers after signing informed consent, placed in citrate-phosphate-dextrose containing adenine (CPDA-1), and centrifuged at 1000 x g for 20 minutes to obtain RBCs. The red blood cells are diluted to a hematocrit of 5% in phosphate-buffered saline (PBS) and centrifuged at 2,000 x g for 10 minutes. This step can be repeated twice to separate the RBCs from plasma proteins. The RBCs are then cross-linked with glutaraldehyde (to form glut-RL RBCs) or with paraformaldehyde and glutaraldehyde (to form para-RL RBCs). Unreacted aldehyde can be removed from the RBCs by centrifugation (this also removes the cells from the plasma proteins), and the cells are finally frozen and lyophilized at -30°C.

Fibrin and fibrinogen can also be purchased from various sources. For example, clinical-grade material is sold by ZLB Behring (Marburg, Germany) under the trade name HAEMOCOMPLETTAN P, and by Baxter (Deerfield, IL USA) under the trade name TISSEEL. Research-grade material can be purchased from Enzyme Research Laboratories (South Bend, IN USA). Fibrin and fibrinogen can also be separated according to methods known in the art (e.g., van Ruijven-Vermeer IA, et al., Hoppe Seylers Z Physiol Chem. 360:633-7 (1979)). Fibrin and fibrinogen can also be separated using glycine, ammonium sulfate, or ethanol precipitation methods known in the art.

RL platelets, RL blood cells, fibrin, or fibrinogen can be in the form of powder, by spraying or blowing dry material added on the matrix and lyophilized. Alternatively, these materials can be added in the matrix as a solution, and dried as described above. In the soaking solution, preservatives such as glycerol, propylene glycol, polyethylene glycol (PEG), trehalose or the like can be included to prevent fabric from becoming brittle or becoming similar to chalk during lyophilization. Generally, the concentration range of preservative in thrombin solution is maximum about 20% (v/v). In a preferred embodiment, the glycerol of about 12% (v/v) is used.

Any combination of RL blood cells, RL platelets, fibrin and/or fibrinogen may be incorporated into the fabric of the present invention. Preferably, the total amount of RL blood cells, RL platelets, fibrin and/or fibrinogen ranges from about 0.1% to about 50% based on the total weight of the dry fabric. In an exemplary embodiment, the hemostatic fabric of the present invention may include the following combination (all weight percentages are expressed based on the total weight of the dry fabric):

In another embodiment, the fabric substrate of the present invention comprises thrombin or a portion comprising thrombin and one or more rehydrated lyophilized (RL) platelets, RL blood cells, fibrin or fibrinogen. For example, a preferred combination of dried platelets, fibrinogen and thrombin is about 3 to 7 wt% RL platelets, 0.75 to 1.5 wt% fibrinogen and 0.1 to 5 wt% thrombin, based on the total weight of the dry fabric. In a particularly preferred embodiment, a combination of about 5 wt% RL platelets, about 1 wt% fibrinogen and about 0.1 wt% thrombin is used.

A hemostatic fabric comprising thrombin and one or more rehydrated lyophilized (RL) platelets, RL blood cells, fibrin or fibrinogen is preferably prepared by the following method: thrombin is incorporated into a matrix, and then one or more rehydrated lyophilized (RL) platelets, RL blood cells, fibrin or fibrinogen are incorporated using the techniques generally described above. In one embodiment, a combination of fibrinogen and thrombin can be injected into the hemostatic fabric of the present invention as described in U.S. 6,113,948, which is incorporated herein by reference. The composition is available from ProFibrix BV (Leiderdorp, The Netherlands) under the trade name "FIBROCAPS" (a combination of fibrinogen microspheres and thrombin microspheres). Preservatives such as glycerol, propylene glycol, polyethylene glycol (PEG), trehalose, etc. can be included in the soaking solution to prevent the fabric from becoming brittle or becoming chalky during lyophilization. Typically, the concentration of the preservative in the thrombin solution ranges up to about 20% (v/v). In a preferred embodiment, about 12% (v/v) glycerol is used.

Generally, the hemostatic fabric of the present invention is prepared by the following steps:

1. Prepare RL platelets or RL blood cells according to the disclosed method and freeze-dry;

2. Prepare hemostatic fabric from the fabric components. During this step, the fabric can be chemically treated, including adding a quantitative reagent such as glycerol, propylene glycol, or polyethylene glycol (PEG) to protect the fabric and facilitate adhesion of the hemostatic protein. Additionally, during this step, serum or plasma containing thrombin can be lyophilized onto the fabric substrate.

3. Apply a hemostatic protein, such as RL platelets, RL blood cells, fibrin, or fibrinogen, directly to the selected surface of the hemostatic fabric (e.g., the surface that contacts the wound tissue) at a preselected particle density (protein/square area of fabric surface) or weight percentage based on the total weight of the fabric. The hemostatic proteins can be applied in any order and can be used in solution or dry form. In one embodiment, RL platelets can be stabilized with an aldehyde and applied to the fabric in a liquid state and then freeze-dried onto the fabric.

4. Package the infused hemostatic fabric and optionally sterilize (eg, gamma or UV radiation).

Detailed examples of hemostatic fabrics and methods of making them are described below.

When the fabric substrate of the present invention is applied to a wound, the hemostatic fabric can activate the body's hemostatic system, including the coagulation system and the vasoconstriction system. It has long been known that various substances can activate platelets and other coagulation factors when in contact with the site of injury. Platelets are the main cellular component of blood that provide hemostasis in response to vascular trauma. When exposed to exogenous substances such as metallic glass and plastic, platelets are activated by contact. See, for example, Barr, H. The stickiness of platelets. Lancet 11, 775 (1941). It is also well known that thrombin converts fibrinogen into fibrin in the coagulation cascade. When applied to a wound, the combination of components in the hemostatic fabric of the present invention works together locally and systemically to activate the coagulation cascade in a highly concentrated and focused manner.

The hemostatic fabric of the present invention can be used as a wound dressing, such as a bandage, gauze, or the like, or can be formed into sutures for surgical use. Other uses include forming the hemostatic fabric of the present invention into a fabric for use in the preparation of linings for protective clothing or clothing, or in tourniquets. In another embodiment, the hemostatic fabric of the present invention is in the form of a kit for use in surgical or first aid or trauma situations. The kit includes a roll, sheet, or other suitable shape of the hemostatic fabric of the present invention, which can be used with or without other blood factors.

Example

The following examples are intended to illustrate, but not to limit, the scope of the present invention. Unless expressly stated otherwise, all parts and percentages are by weight and all temperatures are in degrees Celsius.

substance

The following solutions were used in the following examples.

Anticoagulant Citrate Dextrose (ACD): 0.042 M trisodium citrate, 0.035 M citric acid, 20% (w/v) anhydrous dextrose, pH 4.5.

Citrate-containing saline: 6.2 mM trisodium citrate, 150 mM NaCl, pH 6.5.

Imidazole-buffered saline: 84 mM imidazole, 150 mM NaCl, pH 6.8.

4% paraformaldehyde: _Suspend 20 g paraformaldehyde and 9.4 g _NaH₂PO₄ in 400 ml deionized _H₂O and heat in a water bath to approximately 60°C until dissolved. Set the pH to 7.2 and add water to 500 ml.

Fixative (prepare immediately before use): mix 1 ml ACD, 10 ml 0.135 Molar NaH ₂ PO ₄ , pH=6.5 and 9 ml 4% (w/v) paraformaldehyde.

Imidazole buffer: 84 mM imidazole, pH = 6.8

Citric acid mother solution: 3.2% sodium citrate, pH=7.4

Examples 1-7: Preparation of hemostatic fabric

The following specific fabric combinations were prepared and used in the following experiments:

Fabric 1: Knit; Curved G75 Fiberglass; Fill 30/1 100% Bamboo Rayon OE

The above combination is a fiberglass/bamboo co-woven with "long" oriented (bent) glass fibers (G75, electrical grade E225 spun from E-grade extruded glass) and bamboo fibers "through" the fabric (fill).

Fabric 2: Knit; Curved ECBC150 1/0 1.0Z Fiberglass; Filling: 18/1100% Bamboo Rayon MJS.

Fabric 3: Knit; Curved ECE225 2/0 4.0Z Fiberglass; Fill 18/3100% Bamboo Rayon RS.

Fabric 4: Woven; 1end - ECG75 1/2 fiberglass; Fill 1end - 18/1100% bamboo rayon OE.

Fabric 5: Braided; 2-ply ECG150 1/0 fiberglass twisted with 20/1 100% bamboo rayon.

Fabric 6: Knit; curved ECE225 2/0 4.0Z fiberglass; filling 16/2100% linen.

Fabric 7: Knit; Curved ECH18 1/0 0.7Z fiberglass; Filling 18/2100% Lyocel MJS.

Example 8: Preparation of a hemostatic fabric matrix comprising thrombin

120 mg of angel hair grade glass fiber was mixed with 8 ml of plasma (Innovative Research, Inc., Southfield, MI) and 80 μl of 1 M _CaCl₂ and placed on an oscillator. The mixture was gently mixed on an oscillator for approximately 90 minutes and the glass fiber was separated from the mixture by centrifugation (300 x g for 5 minutes). The supernatant was collected and glycerol was added to a final concentration of 9% by weight. The final product was a serum containing thrombin IIa.

Soak 50 ^cm2 of the above fabric 1 in about 5 ml of the above serum for about 1 minute. Excess serum is drained off and the soaked fabric substrate is frozen at -20°C and lyophilized.

Example 9: Preparation of a hemostatic fabric matrix containing thrombin and an additional hemostatic agent

A. Fabric Matrix of Thrombin and Rehydrated Lyophilized (RL) Platelets

Two methods for preparing a fabric-based hemostatic matrix containing rehydrated lyophilized (RL) platelets and thrombin are detailed below. The amount prepared was estimated using a thrombin generation assay as described below.

1. Method 1

In this method, RL platelets are produced separately and then added to a fabric matrix.

(a) Preparation of RL platelets

RL platelets are prepared as described in U.S. 5,651,966 and 6,139,878, which are incorporated herein by reference. Alternatively, the following method may be used:

Platelet-rich plasma (PRP) is prepared by first drawing fresh venous blood into an ACD (e.g., 42.5 ml of blood into 7.5 ml of ACD in a 50 cc syringe). The blood is centrifuged (10 minutes, 1,200 rpm, at 25-27°C) to separate the red blood cells. PRP is found in the supernatant. Alternatively, PRP can be isolated from stored and/or expired platelets by removing cells from the storage bag and centrifuging at approximately 1200 rpm for 10 minutes to remove contaminating blood cells and aggregated platelets.

Use centrifugation (10 minutes, about 2400rpm) to remove plasma proteins from PRP, and the separated platelets are resuspended in citrate buffer. The platelets are washed twice in citrate buffer and resuspended in citrate buffer to a final concentration of about 8x10 ⁹ platelets/ml. Alternatively, plasma proteins can be removed from PRP by sieve chromatography (Sepharose4B balanced with citrate buffer). The turbid portion can be collected, merged, and centrifuged to separate the platelets. The platelets are then resuspended in citrate buffer to a final concentration of about 8x10 ⁹ platelets/ml.

Crosslinking the isolated platelets involves adding 3.5 ml of fixative solution dropwise to 1.25 ml of isolated platelets at a concentration of approximately 8 x ¹⁰ platelets/ml under gentle stirring. The fixative solution is added to a final volume of approximately 10 ml, and the mixture is incubated at room temperature for 1 hour without stirring or shaking. The crosslinked platelets are centrifuged (10 minutes, 2400 rpm), resuspended in imidazole-buffered saline, washed, and finally resuspended in imidazole-buffered saline to a final concentration of approximately 1 x ¹⁰ platelets/ml.

Cross-linked platelets are frozen and lyophilized by first suspending them in imidazole-buffered saline at pH 6.8 containing 5% bovine serum albumin to a final platelet concentration of approximately 0.8 x ¹⁰⁹ platelets/ml. The mixture is divided into 1 ml aliquots, frozen at -80°C, and lyophilized overnight or longer. The lyophilized product is stored at -20°C. The lyophilized cross-linked platelets can be rehydrated by resuspending them in imidazole buffer.

(b). RL platelets were added to the matrix.

To prepare a matrix containing RL platelets, the RL platelets are first ground into a fine powder in a dry state. The fine powder is evenly sprinkled onto a flat, sterile surface, such as a culture dish, and one side of a freeze-dried, thrombin-loaded matrix prepared as described in Example 8 is pressed onto the RL powder and removed. The powdered RL platelets can also be blown onto fabric using known air blowing techniques. As an alternative, the RL platelets can be prepared without freeze drying and without serum albumin and resuspended in the thrombin serum prepared in Example 8. The desired matrix is then soaked in this solution to obtain the final product. As another alternative, the rehydrated RL platelets can be resuspended in the thrombin serum described in Example 8 and used to soak the desired matrix to obtain the final product.

2. Method 2

In this method, RL platelets are stabilized with aldehydes after being bound to a textile matrix, where they serve as a component of the matrix. The principle is that the platelets are first contact-activated and adhere to the matrix through the normal interaction between hemostatic cells and textile fibers. The platelets and textile matrix are then bonded together through aldehyde stabilization. The method includes the following steps:

(a) Platelet-rich plasma (PRP) is prepared as described above or, alternatively, platelet-rich plasma, typically stored in liquid form, is obtained.

(b) Incubating platelet-rich plasma with an amount of fabric matrix predetermined to bind 90% of the platelets. To achieve the predetermined binding level, a sample of the matrix is incubated with an excess of platelets (more than the amount sufficient to saturate the matrix), and the amount of platelets remaining in the mixture after removal of the matrix is calculated.

(c) The matrix is removed from the PRP and the platelet concentration in the residual fluid is determined.

(d) Incubate the fabric in 10x the volume of citrate water on a shaker for 5 minutes, draining off excess liquid. Repeat this step three times. The soaked fabric substrate is then placed in an appropriate volume of citrate water to ensure that the number of platelets bound to the fabric is 8 x ¹⁰⁹ platelets/ml for the cross-linking step described below.

(e) Add 3.5 ml of fixative solution dropwise to 1.25 ml of platelet-soaked matrix (8 x ¹⁰ platelets/ml) while gently shaking to mix. Add fixative solution more rapidly to further dilute the mixture to a final volume of 10 ml, resulting in a final platelet-bound count of 1 x ¹⁰ platelets/ml. Incubate at room temperature for 1 hour without stirring or shaking. Finally, dilute the platelet-fabric matrix into 10 volumes of imidazole-buffered saline and incubate on a shaker for 5 minutes, draining off excess liquid. Repeat this step three times.

(f) To incorporate thrombin, the platelet-fabric matrix was freed of excess imidazole-buffered saline as thoroughly as possible and then immersed in excess serum IIa as described above. The excess serum IIa was removed and the fabric matrix was frozen and lyophilized as described above.

(g) The lyophilized hemostatic matrix was characterized using a thrombin generation assay (described below).

Those skilled in the art will appreciate that while Examples 8 and 9 utilize Fabric 1 described above to prepare one embodiment of the present invention, any combination of fabrics described herein (e.g., Fabrics 2-7 described above) may alternatively be used.

Thrombin generation assay

The method is based on Fischer, T.H. et al., Synergistic platelet integrin signaling and factor XII activation in poly-N-acetyl glucosamine fiber-mediated hemostasis. Biomaterials 26, 5433-43 (2005). In short, thrombin generation kinetics can be used to reflect the ability of the hemostatic matrix to exert the catalytic surface function of components (e.g., factor XII) of the coagulation cascade. In this analysis, thrombin (IIa) splits the non-fluorescent synthetic substrate peptide -D-Phe-Pro-Arg-ANSNH to produce a fluorescent product. The time course of fluorescence generation is then tracked in a 96-well fluorescent platelet reader in a kinetic model.

96-well plates were blocked overnight at 37°C with 150 μl of 5% BSA and citrate solution and then stored at 4°C until use. A 4 mm diameter (approximately 100 mm) sheet of hemostatic matrix was prepared using a 4 mm Trephine punch, a razor, or sharp scissors. The fluorescent IIa substrate D-Phe-Pro-Arg-ANSNH (Cat #SN-17a-C ₆ H ₁₁ , from Haematologic Technologies, Inc., Essex Junction, VT) was diluted 1/200 into plasma, and CaCl ₂ was added to a final concentration of 10 mM. The mixture was placed in a fluorimeter and fluorescence (490 nm) was measured for approximately 2 hours. Data were analyzed by plotting the time course of each well, measuring the initial and maximum slopes of the relative fluorescence change curves, and the time required to achieve the maximum slope. The initial and maximum slopes and the time to the maximum slope are quality metrics. The higher the slope, the shorter the time to achieve the maximum slope, and the greater the pre-hemostatic properties of the matrix.

Example 10: Preparation of dual fiber fabric

This example illustrates the preparation of a fabric from a combination of glass fibers and another selected textile fiber. Two lines of evidence indicate that continuous, thin glass filaments may be a potential component of a hemostatic fabric. First, it was discovered that platelets activate and adhere to glass (Barr, H. Lancet 238, 609-610 (1941)). As a result, the use of glass tubes for in vitro manipulation of platelets has generally been avoided, and binding to glass has long been a method for evaluating platelet activity (McPherson, J. & Zucker, M.B. Blood 47, 55-67 (1976); Tsukada, T. & Ogawa, T. Rinsho Ketsueki 14, 777-84 (1973); Cooper, R.G., Cornell, C.N., Muhrer, M.E. & Garb, S. Tex Rep Biol Med 27, 955-61 (1969)). Secondly, plasma proteins (Stouffer, J.E. & Lipscomb, H.S. Endocrinology 72, 91-4 (1963); Lissitzky, S., Roques, M. & Benevent, M.T.C R Seances Soc Biol Fil 154, 396-9 (1960); Bull, H.B. Biochim Biophys Acta 19, 464-71 (1956)), FXII (Ratnoff, O.D. & Rosenblum, J.M. Am J Med 25, 160-8 (1958)) and fibrinogen (Sit, P.S. & Marchant, R.E. Thromb Haemost 82, 1053-60 (1999); Rapoza, R.J. & Horbett, T.A.J Biomed Mater Examples of well-studied adsorption processes on external surfaces include those described in [1990] (Res 24, 1263-87; Perez-Luna, V.H., Horbett, T.A. & Ratner, B.D., J Biomed Mater Res 28, 1111-26 (1994)). Chemical and physical adsorption processes can occur on external surfaces (Silberberg, A.J. Physical Chem. 66, 1872-1883 (1962)). FXII (Hagemann factor) was found to be particularly important because it triggers coagulation of body fluids at the glass/blood interface (Ratnoff, supra; Ratnoff, O.D. & Margolius, A., Jr. Trans Assoc Am Physicians 68, 149-54 (1955)). Platelet activation and the reversal of intrinsic coagulation are closely related mechanisms, partly due to the fact that platelets play a catalytic surface role in the assembly of the Va/Xa complex during thrombin generation. Platelet activation by biomaterials (e.g., via integrin outside-in signaling) can lead to the appearance of phosphatidylserine on the surface, an important component of the catalytic complex for thrombin generation (Fischer, T.H., Connolly, R., Thatte, H.S. & Schwaitzberg, S.S. Microsc Res Tech 63, 168-74 (2004)). Factor XII has been found to bind to the platelet surface peripherally to activate the intrinsic coagulation pathway in the cell's microenvironment (Iatridis, P.G., Ferguson, J.H. & Iatridis, S.G. Thromb Diath Haemorrh 11, 355-71 (1964); Shibayama, Y., Reddigari, S. & Kaplan, A.P. Immunopharmacology Vol. 3224-7 (1996)), although the series of proteolytic steps associated with Factor XII occurring on the platelet surface has not been understood. The net effect of the close relationship between Factor XIIa-mediated coagulation and platelet activation is a synergistic effect on the early initiation of fibrin polymerization.

The interaction of liquids with glass is largely governed by surface tension phenomena related to hydrophobicity, zeta potential, and wettability. Liquids have minimal interaction with the interior of the silk. Therefore, we sought another type of fiber with higher adsorption to compensate for the lower liquid transport and glass adsorption. A panel of natural and synthetic fibers was tested for their ability to activate platelets and the intrinsic coagulation cascade. A dual fiber product, comprising a continuous filament of E-glass and a specialized artificial silk, was prepared and tested for its hemostatic effects in a porcine model of bleeding.

E-type continuous filament glass (ECDE 11.6 fiberglass) was provided by Carolina Narrow Fabrics, Inc. (Winston-Salem, NC). Bamboo-derived rayon (Bambusa textilis) and other natural and synthetic fibers were provided by Cheraw Yarn Mills, Inc. (Cheraw, SC). Dual fiberglass/rayon fabric was manufactured by Carolina Narrow Fabrics, Inc. (Winston-Salem, NC). Gauze was from Kendall (Mansfield, MA).

Peripheral blood from consenting healthy volunteers was drawn into citrate anticoagulant and platelet-rich plasma was separated by differential centrifugation as described in detail in Fischer, T.H. et al. Biomaterials 26, 5433-43 (2005). The platelet concentration in the platelet-rich plasma was determined using a Hiska hematology analyzer and the platelet concentration was adjusted to 150,000 platelets/ul by diluting the sample with platelet-free plasma.

The kinetics of thrombin generation were investigated by studying the effects of fibers on the kinetics of thrombin generation in platelet-rich plasma (150,000 platelets/ul). The fibers were hydrolyzed to yield a fluorescent product using the thrombin substrate D-Phe-Pro-Arg-ANSNH. 300 μg of each fiber were tested in triplicate in 100 μl of platelet-rich plasma using the fluorogenic substrate D-Phe-Pro-Arg-ANSNH. The time course of thrombin generation was initiated by adding ₁₀ mM CaCl to each sample. The delay time of thrombin generation was defined as the time point at which fluorescence increased by 10% relative to the initial baseline value.

Thromboelastography Thromboelastography (TEG) measurements were performed using a TEG-5000 Thromboelastography Hemostasis Analyzer (Haemoscope Corporation, Niles, IL). The analysis was initiated by adding _CaCl2 to 10 mM to platelet-rich plasma (150,000 platelets/ul), followed by immediate transfer of 327 ul of calcified platelet-rich plasma to a thromboelastography chamber containing 33 ul of citrate solution. The final fiber concentration was 3.0 mg/ml. Measurements were taken in triplicate at 37°C for 1 hour, and the relevant parameters were extracted from the "hardness" curve.

Scanning electron microscopy SEM analysis of glucosamine-based materials was performed as follows. Whole peripheral blood from a healthy volunteer was flowed directly from a venapuncture butterfly onto a binary-fiber fabric or gauze so that 1 cm x 1 cm of each material was covered with 2 ml of whole blood. The materials were incubated for 1 minute and then diluted to 50 ml with citrate + 1 mM EGTA to stop the hemostasis process. The materials were allowed to settle under gravity for 5 minutes and then diluted again with citrate. This process was repeated two more times to obtain each material without unbound RBCs. One minute after contact with blood, repeated dilutions, and sedimentation of the material/RBC complex, 0.1% (w/v) glutaraldehyde was added and the samples were incubated at room temperature for 1 hour. A 1/1 (v/v) sample was diluted with 4% paraformaldehyde to a final paraformaldehyde concentration of 2%, and then glutaraldehyde was added to a final paraformaldehyde concentration of 0.5%. An initial stabilization step using 0.1% glutaraldehyde has been shown to minimize osmotic pressure-driven changes in the morphology of red blood cells (RBCs) due to exposure to paraformaldehyde (Fischer, T.H. et al. Microsc Res Tech 65, 62-71 (2004)). The samples were stored overnight at 4°C and then examined using a Cambridge S200 scanning electron microscope at 20 kV.

Measurement of bound RBCs 10 mg samples of binary fiber or gauze were directly exposed to 1.0 ml of whole peripheral blood and washed as described for scanning electron microscopy. pGlcNAc was then placed in 10 ml of distilled water diluted with 1% TX-100 to release hemoglobin from bound RBCs. The samples were centrifuged at 10,000 x g for 5 minutes, and the absorbance was measured at 414 nm to quantify the total amount of hemoglobin associated with each substance (and thus the number of RBCs).

Measuring the extent of RBC lysis due to contact with substances As described in the previous two sections, 10 mg samples of dual fiber or gauze were directly exposed to 1.0 ml of whole peripheral blood. After 1 minute of exposure, the samples were centrifuged at 10,000 x g for 5 minutes to precipitate blood cells and other substances. The optical density was measured at 414 nm to quantify the amount of hemoglobin released in the supernatant and, therefore, the amount of blood shed.

Brachial Plexus and Femoral Artery Transverse Hemorrhage Model in Pigs 40 to 50 kg mixed-breed pigs were anesthetized with isoflurane, and several sensors were placed to track hemodynamic and vascular processes: a pulmonary artery thermodilution catheter was inserted into the pulmonary artery via the external jugular vein; a micromanometer-tipped catheter was placed into the right atrium and thoracic aorta via the left femoral vessel; a 22-gauge gauge tube was inserted into the left femoral artery and connected to a suction pump; and a catheter was placed through the left femoral vessel.

The bleeding-inducing phase of the experiment was performed in two stages. First, a therapeutic transsection of the contralateral brachial artery was performed. The brachial artery and two connected veins of ~3 mm diameter were exposed surgically. One side of the artery and two veins was completely incised with a scalpel. The wounds were completed in a nearly simultaneous manner and then immediately wrapped on each side with a dual fiber fabric or gauze. The infiltrated cut site was completely wrapped with each substance and then pressure was applied for 6 minutes. The wrapping was removed and the amount of bleeding was determined as described below. Both wounds were then wrapped again with the dual fiber material to stabilize the animal. The second stage of the experiment was performed by exposing the contralateral femoral artery through the shell. The two contralateral femoral arteries were transected in a nearly simultaneous manner and the surgical site was then wrapped with a dual fiber fabric or gauze. Pressure was applied for 6 minutes and then the material was removed for determination of bleeding.

The amount of blood shed into the original packaging material is determined by placing the binary tissue or gauze in 1 liter of distilled water to lyse the RBCs. After stirring for 2 hours at room temperature and storage at 2°C, the optical density is measured at 414 nm to determine the amount of hemoglobin released, and thus the amount of RBCs shed and the volume of blood lost.

result

The experiments were divided into three phases. First, candidate materials for forming hemostatic fabrics were identified, including measuring the ability of the selected fibers to activate hemostasis in platelet-rich plasma. Second, the dual-fiber combinations were subjected to TEG and SEM analysis to understand the mechanism of function. Finally, the hemostatic ability of the dual-fiber fabrics was evaluated using a porcine bleeding model. Details of these experiments are described below.

The ability of a group of common textile fibers to activate platelets and promote the intrinsic (contact) coagulation pathway was analyzed by candidate fiber activation hemostasis system. Figure 1 depicts the behavior of representative fibers in a fluorescent thrombin generation assay. In Figure 1, two samples of glass, specialized rayon, or gauze fibers were placed in platelet-rich plasma containing a fluorescent thrombin substrate, and then the time course of thrombin generation was triggered by the addition of calcium. The arrows indicate the time of thrombin generation. As shown in Figure 1, exposure of platelet-rich plasma to E-type continuous glass filaments resulted in the generation of thrombin in about 8 minutes. The specialized rayon had lower prothrombogenic activity, with thrombin generation occurring at 12 minutes, while the gauze fibers were much slower.

Figure 2 shows the behavior of the fibers in the more swellable group. In Figure 2, the designated fibers were tested as described in Figure 1 to determine the thrombin generation time. Error bars represent the standard deviation of duplicate analyses. As shown in Figure 2, glass and specialty rayon were the first and second best thrombogenic materials tested, respectively. Chitin and gauze are components of surface hemostatic products that do not strongly promote thrombin generation. Therefore, a prototype bandage was constructed from glass and specialty rayon.

The in vitro properties of the glass/specialty rayon dual-fiber fabric are shown in Figure 3 , comparing the dual-fiber matrix and gauze in a thromboelastography analysis using platelet-rich plasma. In Figure 3 , the dual-fiber or yarn was placed in a thromboelastography tube containing normal saline, followed by the addition of platelet-rich plasma and calcium to initiate a time course of clot formation. A normal saline solution without the substance was run as a negative control. As shown in Figure 3 , the glass/specialty rayon fabric significantly promoted fibrin clot formation compared to the gauze or saline control. As shown in Figure 4 , the dual-fiber matrix and gauze were analyzed by scanning electron microscopy after exposure to excess peripheral blood. In Figure 4 , the dual-fiber or gauze were saturated with excess peripheral blood and then examined by scanning electron microscopy as described above. The white arrows in the left two dual-fiber groups indicate the specialty rayon fibers. As shown in Figure 4 , the glass/specialty rayon matrix tightly bound a large number of RBCs, while these cells were only sparsely covered on the gauze matrix.

Accompanying drawing 5 shows the amount of RBCs number on each matrix. In Accompanying drawing 5, dual fiber or gauze is saturated with excessive peripheral blood, and then the amount of bound RBCs is measured as described above. Error bars represent the standard deviation of duplicate determinations. As shown in Accompanying drawing 5, the RBCs bound by dual fiber fabric are 10 times that of gauze. No significant cell lysis of RBCs occurs (data not shown). SEM inspection of dual fabric matrix also shows that a large amount of highly activated platelets are arranged on the continuous glass fiber component, as shown in Accompanying drawing 6, which shows that the dual fabric is saturated, washed with peripheral blood, and examined with a scanning electron microscope. These results show that in terms of surface hemostasis, glass/specialized rayon matrix is more effective than gauze.

The ability of the dual fiber bandage to stop bleeding in the pig model is compared with gauze in severe pig large vessel transect injuries. Two types of injuries were determined in each of four pigs. First, the brachial artery and two connected large veins in the contralateral plexus area were completely transected in a nearly simultaneous manner. This resulted in natural arterial and venous exsanguination. The contralateral incision/injury site was immediately wrapped with the dual fiber fabric or gauze required to fill the injury site. The pressure was then kept on for 6 minutes, and the package was then removed from the site. The degree of hemostasis was determined to be complete (no visible bleeding), partial (less than 3 ml of blood loss per minute) or uncontrolled (more than 3 ml of blood loss per minute). Blood loss and any bleeding on the packaging material were measured from each wound site. The contralateral brachial plexus injury site was then wrapped again with the dual fiber fabric to stabilize the animal for the second group of femoral injuries. The contralateral femoral artery was exposed and then completely transected in a nearly simultaneous manner to induce exsanguination. If the brachial plexus was injured, the injury site was immediately wrapped with the dual fiber fabric or gauze. After 6 minutes of continuous pressure, the wrap was removed from the site and the degree of hemostasis was determined as described for the brachial injury. An important feature of this large vessel transection model is that the animals were not in hemorrhagic shock. Since the wound was immediately wrapped under pressure, the mean arterial pressure was maintained in the range of 45 to 55 mmHg and the total blood loss did not exceed 5% of the total blood volume. As shown in Figure 7, in the brachial plexus and thigh injuries, the total amount of blood loss from the dual fabric material was about half that of the gauze. In Figure 7, the total amount of material adsorption and bleeding during the six-minute compression period was measured from the arm (left panel A) and thigh (right panel B). The error bars represent the standard deviation of blood loss from similar injuries in 5 animals. As shown in Figure 7, the gauze had a clear tendency to tear the hemostatic plug (to the extent of one), while the dual fiber fabric did not strongly incorporate into the hemostatic zone.

These results demonstrate that the fundamental principles of hemostasis can be used to design affordable materials for surface hemostasis. When the fiber components of the fabric were optimized for thrombogenicity, the glass/specialized rayon fabric outperformed gauze in capillary and large vessel injuries in a porcine model; both bleeding time and blood loss were reduced by approximately half.

Although the present invention has been described above with reference to specific embodiments, it is apparent that many changes, modifications and variations can be made without departing from the scope of the invention disclosed herein. Therefore, it is intended to include all such changes, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims (35)

1. A hemostatic fabric, comprising: A sterile material comprising a combination of continuous glass fiber filaments and bamboo rayon fibers, wherein the sterile material is contained in a package, wherein the continuous glass fiber filaments have a diameter of 5 nanometers to 15 micrometers, and wherein the continuous glass fiber filaments comprise aluminosilicate glass. Thrombin or a portion containing thrombin; and One or more hemostatic agents, including one or more of rehydrated low-pressure lyophilized platelets, rehydrated low-pressure lyophilized blood cells, fibrin and fibrinogen; When applied to wounds, the hemostatic fabric can activate the body's hemostatic system. The relative amounts of glass fiber and bamboo rayon fiber, based on the total weight of the fabric, range from 50% to 80% by weight of glass fiber and 50% to 20% by weight of bamboo rayon fiber. 2. The hemostatic fabric of claim 1 in knitted or non-knitted form. 3. The hemostatic fabric of claim 1, wherein the hemostatic fabric is a cover or a lining of a cover. 4. The hemostatic fabric of claim 1, wherein the sterile material comprises 65% by weight of glass fiber and 35% by weight of bamboo rayon fiber. 5. The hemostatic fabric of claim 1, wherein the portion containing thrombin further comprises one or more coagulation factors selected from factor XII, factor XIIa, factor XI, factor XIa, factor XIII, factor XIIIa, factor IX, factor IXa, factor VIII, factor VIIIa, factor VWF, factor V, factor Va, factor X, factor Xa, and combinations thereof. 6. The hemostatic fabric of claim 1, wherein said portion further comprises a vasoactive agent. 7. The hemostatic fabric of claim 6, wherein the vasoactive agent is selected from endothelin, thromboxane, NO scavenger, and combinations thereof. 8. The hemostatic fabric of claim 1 further comprises a preservative selected from glycerin, propylene glycol, polyethylene glycol, trehalose, and combinations thereof. 9. The hemostatic fabric of claim 1, wherein the portion containing thrombin is a partially purified plasma component containing thrombin IIa. 10. The hemostatic fabric of claim 1, wherein the thrombin or the portion containing thrombin is 0.01 to 10% by weight based on the total weight of the fabric. 11. The hemostatic fabric of claim 1, wherein the thrombin or the portion containing thrombin is 0.05 to 7% by weight based on the total weight of the fabric. 12. The hemostatic fabric of claim 1, wherein the thrombin or the portion containing thrombin is 0.1 to 5% by weight based on the total weight of the fabric. 13. The hemostatic fabric of claim 1 further comprises one or more other agents selected from skin modifiers, anti-inflammatory agents, analgesics, antibacterial agents, vasoconstrictors, growth factors, anti-scarring agents, burn agents, dehydrating agents, antithrombotic agents, and combinations thereof. 14. The hemostatic fabric of claim 13, wherein the antimicrobial agent comprises an antifungal agent. 15. The hemostatic fabric of claim 1, wherein the rehydrated low-pressure lyophilized platelets or the rehydrated low-pressure lyophilized blood cells account for 0.1 to 20 wt% based on the total weight of the fabric. 16. The hemostatic fabric of claim 1, wherein the fibrin or fibrinogen accounts for 0.1 to 5 wt% based on the total weight of the fabric. 17. A method for preparing the hemostatic fabric according to claim 1, comprising the following steps: A substance comprising a combination of continuous glass fiber filaments and bamboo rayon fibers is contacted with thrombin or a portion comprising thrombin and a hemostatic agent selected from rehydrated low-pressure lyophilized platelets, rehydrated low-pressure lyophilized blood cells, fibrin, fibrinogen, and combinations thereof to form a moist matrix, wherein the continuous glass fiber filaments have a diameter of 5 nanometers to 15 micrometers, and wherein the continuous glass fiber filaments comprise aluminosilicate glass; and The moistened matrix is dried to form the hemostatic fabric. 18. The method of claim 17, wherein the portion further comprises one or more coagulation factors selected from factor XII, factor XIIa, factor XI, factor XIa, factor XIII, factor XIIIa, factor IX, factor IXa, factor VIII, factor VIIIa, factor VWF, factor V, factor Va, factor X, factor Xa, and combinations thereof. 19. The method of claim 17, wherein the portion further comprises a vasoactive agent. 20. The method of claim 19, wherein the vasoactive agent is selected from endothelin, thromboxane, NO scavengers, and combinations thereof. 21. The method of claim 17, wherein the fabric further comprises a preservative selected from glycerin, propylene glycol, polyethylene glycol, trehalose, and combinations thereof. 22. The method of claim 17, wherein the thrombin-containing portion is a partially purified plasma component containing thrombin IIa. 23. The method of claim 17, wherein the fabric further comprises one or more other agents selected from skin modifiers, anti-inflammatory agents, analgesics, antibacterial agents, vasoconstrictors, growth factors, anti-scarring agents, burn agents, dehydrating agents, antithrombotic agents, and combinations thereof. 24. The hemostatic fabric of claim 23, wherein the antimicrobial agent comprises an antifungal agent. 25. The method of claim 17, wherein the material is in a knitted or non-knitted form. 26. A method for preparing the hemostatic fabric according to claim 1, comprising the following steps: A material comprising a combination of continuous glass fiber filaments and bamboo rayon fibers is contacted with platelet-rich plasma containing platelets, wherein the continuous glass fiber filaments have a diameter of 5 nanometers to 15 micrometers, and wherein the continuous glass fiber filaments comprise aluminosilicate glass. Cross-linking the platelets; and The fabric is brought into contact with thrombin or a portion containing thrombin to form the hemostatic fabric. The platelets mentioned therein are rehydrated, low-pressure freeze-dried platelets. 27. The method of claim 26 further comprises the step of drying the hemostatic fabric. 28. The method of claim 26, wherein the portion further comprises one or more coagulation factors selected from factor XII, factor XIIa, factor XI, factor XIa, factor XIII, factor XIIIa, factor IX, factor IXa, factor VIII, factor VIIIa, factor VWF, factor V, factor Va, factor X, factor Xa, and combinations thereof. 29. The method of claim 26, wherein the portion further comprises a vasoactive agent. 30. The method of claim 29, wherein the vasoactive agent is selected from endothelin, thromboxane, NO scavengers, and combinations thereof. 31. The method of claim 26, wherein the fabric further comprises a preservative selected from glycerin, propylene glycol, polyethylene glycol, trehalose, and combinations thereof. 32. The method of claim 26, wherein the thrombin-containing portion is a partially purified plasma component containing thrombin IIa. 33. The method of claim 26, wherein the fabric further comprises one or more other agents selected from skin conditioners, anti-inflammatory agents, analgesics, antibacterial agents, vasoconstrictors, growth factors, anti-scarring agents, burn agents, dehydrating agents, antithrombotic agents, and combinations thereof. 34. The hemostatic fabric of claim 33, wherein the antimicrobial agent comprises an antifungal agent. 35. The method of claim 26, wherein the material is in a knitted or non-knitted form.