HK1136765B

HK1136765B - Process for production of solid wound dressing

Info

Publication number: HK1136765B
Application number: HK10102098.5A
Authority: HK
Inventors: M‧马科皮; D‧伯尔
Original assignee: Stb有限公司
Priority date: 2006-08-04
Filing date: 2007-08-06
Publication date: 2014-05-09

Description

Method for producing solid wound dressing

Technical Field

The present invention relates to a method of producing a solid dressing for treating wounded tissue in a mammalian patient, such as a human, and to the dressing and intermediate products produced thereby.

Invention backLandscape

Materials and methods (gauze dressing, directed pressure, and hemostat) that can be used for pre-hospitalization care to stop bleeding have unfortunately not changed significantly over the past 2000 years. See L.Zimmerman et al, Great Ideas in the History of Surgery (SanFrancisco, Calif.: Norman Publishing; 1993), 31. Even for trained people, they are not as effective, and it is not uncommon for a large amount of bleeding or fatal blood loss to occur at an accessible site. See j.m. rocko et al, j.trauma 22: 635(1982).

Mortality data from vietnam show that 10% of war deaths are due to uncontrolled extreme blood loss. See SAS/STAT Users Guide, 4th ed. (Cary, N.C.: SASInstitute Inc.; 1990). At most one third of deaths due to bleeding during the Vietnam War (Vietnam War) can be prevented using effective field blood loss control methods. See SAS/STAT Users Guide, 4th ed. (Cary, N.C.: SAS institute Inc; 1990).

Although civilian trauma mortality statistics do not provide an exact number of pre-hospitalized deaths due to extreme blood loss, case and anecdotal reports are shown as the same. See j.m. rocko et al. These data show that a substantial increase in survival can be achieved by using a simple and effective method of blood loss control prior to hospitalization.

Many newer hemostatic agents are now in use that have been developed to overcome the drawbacks of conventional gauze bandages. These hemostatic agents include the following: microporous polysaccharide particles (Trauma)Medafor Inc.，Minneapolis，

MN); zeolite (Z-medical Corp, Wallington, CT); acetylated poly-N-acetylglucosamine (Rapid)Depolyenzyme Hemostat (RDH), Marine Polymer Technologies, Danvers, MA); chitosan: (A)bandage, HemCon medical technologies inc., Portland OR); liquid fibrin sealant (Tisseel VH, Baxter, Deerfield, EL). Thrombin on human fibrinogen and equine collagen (Tachocomb-S, Hafslundnycomed Pharma, Linz, Austria); microdispersed oxidized cellulose (m doc, allracel Group, Dublin, Ireland); propyl pernate (Hemostatin, Analytical Control Systems inc., Fishers, EN); epsilon-aminocaproic acid and thrombin (Hemarrest patch, clarion pharmaceuticals, Inc); purified bovine dermal collagen (A)Sheet material (nonwoven web or avitene Microfiber Collagen Hemostat (MCH), Davol, Inc., Cranston, RI); controlled oxidation of regenerated cellulose (Ediicon inc., Somerville, NJ); aluminum sulfate with an ethylcellulose coating (Sorbastace Microcaps, Hemospace, LLC, New Or leaves, LA); microporous hydrogel-forming polyacrylamides (BioHemostat, Hemodyne, inc., Richmond VA); and recombinant activated factor VII (NovoNordisk inc, Princeton, NJ). These agents have met with varying degrees of success in animal models for traumatic injury and/or when used in the field.

One such agent is a starch-based hemostatic agent sold under the trade name TraumaDEX. Such products include microporous polysaccharide particles that are poured directly into or onto the wound. The particles appear to exert their hemostatic effect by absorbing water from the blood and plasma in the wound causing the accumulation and enrichment of clotting factors and platelets. However, in both studies of the lethal groin wound model, this agent did not show meaningful benefits over standard gauze dressings. See McManus et al, Business Briefing: emergencymedical Review 2005, pp.76-79 (currently available online at the website of www.touchbriefings.com/pdf/1334/Wedmore. pdf).

Another particle-based agent is QuickClot powder, zeolite granular hemostat for pouring directly into or onto wounds. Zeolite particles also appear to exert their hemostatic effects by causing the accumulation and enrichment of clotting factors and platelets through fluid absorption. Although such agents have been used successfully in certain animal studies, there are concerns about the exothermic process upon absorption of liquid by the particles. Some studies have shown that this reaction produces temperatures in excess of 143 ℃ in vitro and temperatures in excess of 50 ℃ in vivo, which is sufficient to cause third degree burns. See McManus et al, Business Briefing: emergenecy Medical Review 2005, at 77. It has also been observed that the exothermic reaction of QuikClot causes macroscopic and histological changes of unknown clinical importance. Acheson et al, j. trauma 59: 865-874(2005).

Unlike these particle-based agents, Rapid delivery Hemostat appears to exert its hemostatic effects through erythrocyte aggregation, platelet activation, activation of the coagulation cascade, and local vasoconstriction. Rapid delivery Hemostat^TMIs a dressing made of poly-N-acetylglucosamine and derived from algae. While the original dressing design was effective in reducing minor bleeding, the addition of a gauze pad was required to reduce blood loss in a swine model of aortic and liver injury. See McManus et al, Business Briefing: emergenecy Medical Review 2005, at 78.

Another dressing derived from poly-N-acetylglucosamine is the hemcon chitosan band, which is purportedly a freeze-dried chitosan dressing designed to optimize mucoadhesive surface density and structural integrity of chitosan at the wound site. HemCon Chitosan band apparently exerts its hemostatic effect primarily through adhesion to the wound, although evidence suggests that it may also enhance platelet function and incorporate red blood cells into the clot formed on the wound. This bandage showed improved hemostasis and less blood loss in several animal models of arterial bleeding, but significant variability was observed between different bandages, including some that failed due to insufficient adhesion to the wound. See McManus et al, Business Briefing: emergenecy Medical Review 2005, at 79.

Liquid fibrin sealants, such as Tisseel VH, have been used for years as operating room accessories for blood loss control. See j.l.garza et al, j.trauma30: 512-513 (1990); kram et al, j. trauma 30: 97-101 (1990); ochsner et al, j. trauma 30: 884 + 887 (1990); t.l.matthew et al, ann.thorac.surg.50: 40-44 (1990); h.jakob et al, j.vase.surg., 1: 171-180(1984). The first appearance of tissue glue for hemostasis is traced back to 1909. See Current Trends in Surgical Tissue Adhesives: proceedings of the First International Symposium on scientific Adhesives, M.J. MacPhee et al, eds. (Lancaster, Pa.: technomic publishing Co; 1995). Liquid fibrin sealant typically consists of fibrinogen and thrombin, but may also comprise factor XIII/XIIIa, either as a by-product of the purification of the fibrinogen or as an added component (thus in certain applications it is not necessary that factor XIII/factor XIIIa be present in the fibrin sealant, since sufficient factor XIII/XIIIa or other transaminase is present endogenously to induce fibrin formation). However, as a liquid, these fibrin sealants have not proven useful for treating traumatic injuries in the field.

Dried fibrinogen-thrombin dressings with collagen carriers (e.g. tachcomb, tachcomb H and tachcosil available from haemophil and nymed Pharma, Linz, Austria) are also used in many european countries for operating room applications. See U.S. Schiiele et al, Clin. materials 9: 169-177(1992). Although these fibrinogen-thrombin dressings do not require the pre-mixing required for liquid fibrin sealants, their utility for field applications is limited by storage conditions of 4 ℃ and the need to be pre-wetted with saline solution prior to application to the wound. These dressings are not very effective for high pressure, massive bleeding. See sonden et al, j. trauma54: 280-285(2003).

A dry fibrinogen/thrombin dressing for treating wounded tissue is also available from the American Red Cross (ARC) as disclosed in us patent 6,762,336, this particular dressing consisting of a backing material and a plurality of layers, the outermost two layers of which comprise fibrinogen (but no thrombin) and the inner layer comprises thrombin and calcium chloride (but no fibrinogen). Although this dressing has been able to show great success in several animal models of blood loss, the bandage is fragile, inflexible, and has a tendency to split when handled. See McManus et al, BusinessBriefing: emergency Medical Review 2005, at 78; kheirabadi et al, j. trauma 59: 25-35(2005).

Other fibrinogen/thrombin based dressings have also been proposed. For example, U.S. patent 4,683,142 discloses a resorbable sheet for use in closing and repairing wounds that is composed of a glycoprotein matrix (e.g., collagen) that includes clotting proteins (e.g., fibrinogen and thrombin. U.S. patent 5,702,715 discloses a reinforced biological sealant composed of separate layers of fibrinogen and thrombin, at least one of which further includes a reinforcing filler, such as PEG, PVP, BSA, mannitol, FICOLL, dextran, inositol, or sodium chlorate. Us patent 6,056,970 discloses a dressing consisting of a bioabsorbable polymer (e.g. hyaluronic acid or carboxymethylcellulose) and a hemostatic composition consisting of powdered thrombin and/or powdered fibrinogen. Us patent 7,189,410 discloses a bandage consisting of a backing material having thereon: (i) fibrinogen particles; (ii) thrombin particles; and (iii) calcium chloride. U.S. patent application publication US 2006/0155234A 1 discloses a dressing consisting of a backing material and a plurality of fibrinogen layers having discrete zones of thrombin therebetween. To date, none of these dressings have been approved for use or are commercially available.

In addition, past efforts to make fibrinogen/thrombin solid dressings have always been hampered by the very nature of their being desirable components for treating wounds, i.e., their inherent ability to react rapidly to form fibrin under aqueous conditions. The presence of factor XIII causes the mixture to cause further conversion of fibrin Ia to cross-linked fibrin II.

The overall clotting process for humans is shown in figure 1. As described therein, the conversion of fibrinogen to fibrin I involves cleavage of two small peptides (a and B) from the α and β chains of fibrinogen, respectively. These small peptides are difficult to detect and monitor directly; however, the decrease in molecular weight of the α and β chains resulting from this cleavage can be monitored by gel electrophoresis. Likewise, the conversion of fibrin I to cross-linked fibrin II can be followed by the disappearance of the gamma chain monomer of fibrinogen on the gel (which is converted to gamma-gamma dimer with the action of factor XIII on the gamma chain monomer).

To avoid premature reactions, previous efforts to produce fibrinogen/thrombin solid dressings have emphasized separating the fibrinogen and thrombin components as much as possible to avoid them forming too much fibrin prior to use of the dressing. For example, fibrinogen-thrombin dressings with collagen carriers (e.g., tachcocomb H, and tachcosil) available from Hafslund nyomedpharma are prepared by suspending fibrinogen and thrombin particles in a non-aqueous liquid, and then spraying the suspension onto a collagen matrix. The use of a non-aqueous environment, as opposed to an aqueous environment, is intended to prevent excessive interaction between fibrinogen and thrombin.

Alternatives to this process have been proposed, each of which is designed to retain fibrinogen and thrombin as separately as possible. For example, the fibrinogen/thrombin solid dressing disclosed in us patent 7,189,410 was prepared as follows: the powdered fibrinogen and powdered thrombin are mixed without any solvent and the dry powder mixture is then applied to the adhesive side of the backing material. The fibrinogen/thrombin solid dressings disclosed in U.S. patent 6,762,336 and U.S. patent application publication US2006/0155234 a1 comprise separate and discrete layers of fibrinogen or thrombin, each substantially free of another component. However, these methods have not been completely successful.

In order to function properly, fibrinogen/thrombin based solid dressings must meet several criteria. First, fibrinogen and thrombin must interact successfully to form a clot, and the more adherent such a clot to a wound, the better the dressing. In general, dressings must have a high degree of integrity, since the loss of active ingredient due to cracking, flaking, etc. ultimately leads to reduced performance and poor user acceptance. Known fibrinogen/thrombin solid dressings have been reported to lack one or more of these characteristics.

Furthermore, the dressing must be homogenous, since all areas of the dressing must function equally well in order to ensure its successful application. The dressing must also hydrate quickly without significant or specialized effort. Relatively flat dressings are often preferred, as it is possible to avoid curling or irregular non-planar structures (which tend to contradict effective application and may in some cases lead to poor performance). Flexibility is another feature that is highly preferred, both to improve performance and to increase the number of geometries and locations of wounds that can be actually treated. Although known fibrinogen/thrombin solid dressings may be soft when hydrated, they do not have sufficient moisture content to be soft prior to hydration. See, e.g., sonden et al, j. trauma 54: 280-285 (2003)); holcomb et al, J.Trauma, 55518-526; McManus & Wedmore, Emergeney Medicine Review, pp76-79, 2005.

The amount of fibrin (in particular insoluble cross-linked fibrin II) present in the dressing prior to use must be relatively small. This latter feature is important for several reasons. First, the presence of insoluble fibrin during the manufacturing process often results in poor quality dressings that may exhibit reduced integrity, lack of homogeneity, and difficulty/slowness of hydration. These results can generally be visually detected by those skilled in the art.

For example, the presence of pre-formed fibrin in a fibrinogen/thrombin based solid dressing may be visually detected by a lack of homogeneity surface appearance. In particular, a rough or rugged appearance often suggests that significant amounts of fibrin are formed during production and may impede future performance. Firm, smooth & shiny "sheets" on the surface of solid dressings are also signs that fibrin tends to hydrate slowly (even blocking) during use. Excessive upward curling of the solid dressing is another indication that significant amounts of fibrin were formed during the manufacturing process. Dressings with excessive fibrin content slowly hydrate and often require the forceful application of liquid, sometimes requiring mechanical penetration of the surface, to initiate hydration when water or aqueous solutions are added. In addition, dressings with high amounts of pre-formed fibrin often have a mottled color mixing patch and a significantly heterogeneous appearance after hydration.

The amount of preformed fibrin may also be evaluated by various biochemical tests, such as the method described in U.S. patent application publication No. US2006/0155234 a 1. According to this test, the conversion of fibrinogen gamma chains to cross-linked gamma-gamma dimers serves as an indication of the presence of fibrin (the proportion of gamma chains converted to gamma-gamma dimers is a measure of the amount of fibrin produced).

Other assays may evaluate changes in the chains of other components of fibrinogen, such as conversion of the a α chain to free α chain and fibrinopeptide a, or conversion of the B β chain to free β chain and fibrinopeptide B. These changes can be monitored by gel electrophoresis in a similar manner as the conversion of gamma to gamma as described in U.S. patent application publication US 2006/0155234A 1. Interestingly, higher levels of γ - γ dimerization (up to 10%) were reported in U.S. patent application publication US2006/0155234 a1, showing that these dressings include significant amounts of fibrin prior to use. This observation may explain the delamination and/or cracking problems observed in some of these dressings.

For a properly functioning fibrinogen/thrombin based solid dressing, hydration is usually complete within a few seconds and only water (or some aqueous solution) needs to be applied over the dressing. This solution may be blood or another body fluid from the site of injury at which the dressing is used, or it may come from some external source, such as saline or other physiologically acceptable aqueous solution applied to the dressing while it is on the wound to be treated. Longer hydration times, i.e., typically greater than 5 seconds, can interfere with the performance of the dressing because the dressing may run off or fall out into the fluid, which continues to be free flowing before fully cross-linked fibrin is formed. Given the potentially fatal consequences of continued bleeding, any delay in hydration of the dressing during use is highly undesirable. In addition, the performance of dressings with excessive fibrin content is often poor, as reflected by the EVPA and adhesion tests described herein, as well as the reduction in scores during in vivo testing and clinical application.

Accordingly, there remains a need in the art for solid dressings that can be used to treat wounded tissue, particularly for wounded tissue in the field due to traumatic injury.

Disclosure of Invention

It is therefore an object of the present invention to provide a method of producing a solid dressing which can treat wounded mammalian tissue, in particular for treating wounded tissue due to traumatic injury. It is a further object of the present invention to provide a solid dressing for treating wounded mammalian tissue, in particular human tissue, and an intermediate product for producing such a dressing. Additional objects, features and advantages of the invention will be set forth in the detailed description which follows, and in part will be obvious from the description, and/or may be learned by practice of the invention. These objects and advantages are achieved and attained by the methods and compositions described in this specification and particularly pointed out in the claims that follow.

In accordance with these and other objects, a first embodiment of the present invention is directed to a method of producing a solid dressing for treating wounded tissue in a mammal, comprising: (a) forming a liquid aqueous mixture of a fibrinogen component and a fibrinogen activator at a temperature sufficiently low to inhibit activation of the fibrinogen component by the fibrinogen activator; (b) reducing the temperature of the aqueous mixture to form a frozen aqueous mixture; and (c) reducing the moisture content of the frozen aqueous mixture to produce a solid dressing having a haemostatic layer consisting essentially of a fibrinogen component and a fibrinogen activator.

Other embodiments of the invention relate to solid dressings produced by this method and intermediate products produced during this process.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation, but not limitation, of the invention as claimed herein.

Brief Description of Drawings

FIG. 1 is a review of the human coagulation cascade provided by the ERL website (http:// www.enzymeresearch.co.uk/coag. htm).

Fig. 2 is a schematic of the equipment used for the ex vivo porcine arteriotomy test described herein.

FIGS. 3A to 3C are graphs showing the results obtained in example 1.

Fig. 4A and 4B are graphs depicting the results of EVPA and adhesion tests performed on the dressings prepared in examples 6-12.

FIGS. 5A and 5B are graphs of the performance characteristics of the frozen compositions shown in example 13 stored at-80 ℃.

FIGS. 6A-6D and 7A-7B show the results achieved in examples 20, 21 and 22.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned herein are incorporated by reference.

As used herein, the use of a singular article, such as "a," "an," and "the" is intended to exclude the plural forms of the subject matter of the article, unless the context clearly and clearly dictates otherwise.

As used herein, "patient" refers to a human or animal individual in need of medical care and/or treatment.

As used herein, "trauma" refers to any damage to any tissue of a patient that results in the loss of blood from the circulatory system and/or the loss of any other fluid from the patient's body. The tissue may be an internal tissue, such as an organ or a blood vessel, or an external tissue, such as skin. The loss of blood may be from the inside, e.g. from a ruptured organ, or from the outside, e.g. from a tear. The wound may be in soft tissue, such as an organ, or in hard tissue, such as bone. The damage may be caused by any agent or source, including traumatic injury, infection, or surgery.

As used herein, "absorbable material" refers to a material that naturally breaks down in and/or by the mammalian body into components that are consumed or eliminated so as not to significantly interfere with wound healing and/or tissue regeneration, and not to cause any significant metabolic disturbance.

As used herein, "stability" refers to the maintenance of those characteristics that determine the activity and/or function of a material.

As used herein, "suitable" refers to a material that does not adversely affect the stability of the dressing or any of its components.

As used herein, "adhesive" refers to a compound or mixture of compounds that improves the adhesion and/or cohesion of the components of the haemostatic layer of the dressing.

As used herein, "solubilizing agent" refers to a compound or mixture of compounds that improves the dissolution of one or more proteins in an aqueous solvent.

As used herein, "filler" refers to a compound or mixture of compounds that provides volume (bulk) and/or porosity to the haemostatic layer of the dressing.

As used herein, "release agent" refers to a compound or mixture of compounds that facilitates removal of the dressing from the production mold.

As used herein, "blowing agent" refers to a compound or mixture of compounds that generates a gas upon hydration under suitable conditions.

As used herein, "solid" means that the shape or form of the dressing does not change significantly when the dressing is placed on a rigid surface, facing down towards the wound, and then left to stand at room temperature for 24 hours.

As used herein, "frozen" means that the composition does not substantially change shape or form when placed on a rigid surface with the wound side down and then left to stand at-40 ℃ for 24 hours, but the composition changes significantly when placed on a rigid surface with the wound side down and then left to stand at room temperature for 24 hours. Thus, in the context of the present invention, a "solid" dressing is different from "frozen" and a "frozen" composition or mixture is different from "solid".

A first preferred embodiment of the present invention relates to a method of producing a solid dressing for treating wounded tissue in a mammal, comprising: (a) forming a liquid aqueous mixture of a fibrinogen component and a fibrinogen activator at a temperature sufficiently low to inhibit activation of the fibrinogen component by the fibrinogen activator; (b) reducing the temperature of the aqueous mixture to form a frozen aqueous mixture; and (c) reducing the moisture content of the frozen aqueous mixture to produce a solid dressing having a haemostatic layer consisting essentially of a fibrinogen component and a fibrinogen activator.

As used herein, "consisting essentially of means that the fibrinogen component and fibrinogen activator are essential and necessary components of the haemostatic layer of the solid dressing when the solid dressing is intended to be used to treat wounded tissue. Thus, for a particular application, the haemostatic layer may contain other components in addition to the fibrinogen component and the fibrinogen activator as required, but these other components are not required for the intended function under normal circumstances, i.e. they are not necessary for the fibrinogen component and the fibrinogen activator to react and form sufficient fibrin to reduce blood and/or fluid egress from normal wounded tissue when the dressing is applied to tissue under the intended application conditions. However, if the conditions of use in a particular situation are not normal, e.g. the patient is a hemophilia patient with a deficiency in factor XII1, appropriate additional components, e.g. factor X111/XIIIa or some other transaminase, may be added to the haemostat layer without departing from the spirit of the invention. Likewise, the solid dressing of the present invention may comprise one or more of these haemostatic layers as well as one or more other layers such as one or more support layers (e.g. backing material or internal support material) and an anti-adhesive layer.

Other preferred embodiments of the present invention relate to methods of treating wounded tissue in a mammal comprising placing a solid dressing of the present invention on the wounded tissue and applying sufficient pressure to the dressing for a time sufficient to form sufficient fibrin to reduce the loss of blood and/or other fluids from the wound.

Other preferred embodiments relate to compositions consisting essentially of a mixture of a fibrinogen component, a fibrinogen activator, and water, wherein these compositions are stable at reduced temperatures for at least 24 hours. These include frozen compositions and liquid compositions. Such compositions are particularly useful for preparing the haemostatic layer of the solid dressing of the invention, but may also be used by themselves to treat wounded tissue.

According to certain embodiments of the invention, the haemostatic layer of the solid dressing is formed or cast as a unitary body. According to these embodiments, the haemostatic layer may be formed by introducing an aqueous solution of the fibrinogen component and an aqueous solution of the fibrinogen activator into a suitable container, such as a mould or the like, with mixing. The resulting aqueous mixture is then cooled to form a frozen aqueous mixture of the fibrinogen component and the fibrinogen activator.

According to certain other embodiments of the invention, the haemostatic layer is formed from a single source, such as an aqueous solution comprising a mixture of fibrinogen and a fibrinogen activator. According to these embodiments, the haemostat layer may be formed as follows: the liquid aqueous mixture of fibrinogen component and fibrinogen activator is introduced into a suitable container, such as a mold or the like, and the temperature is then reduced to form a frozen aqueous mixture of fibrinogen component and fibrinogen activator.

Preferably, in each of these embodiments of the invention, the haemostat layer is preferably substantially homogeneous throughout.

According to certain preferred embodiments of the present invention, the solid dressing is produced using a mould. According to these embodiments, the solid dressing may optionally further comprise an anti-adhesion layer in addition to the haemostatic layer and the support layer. As used herein, a "release layer" refers to a layer comprising one or more agents ("release agents") that facilitate or aid in the removal of a solid dressing from a mold in which the solid dressing has been produced. A preferred such agent is sucrose, but other suitable release agents include gelatin, hyaluronidase and its derivatives (e.g. hyaluronic acid), mannitol, sorbitol and glucose. Such a release layer is preferably arranged or formed in the mould prior to introducing the liquid aqueous mixture or solution of the fibrinogen component and the fibrinogen activator.

Alternatively, one or more such release agents may be included in the haemostat layer. According to these embodiments, the release agent may be introduced into the liquid aqueous mixture prior to or during introduction of the liquid aqueous mixture into the mold. A release agent may also be introduced into the solution of fibrinogen component and/or the solution of fibrinogen activator prior to or during formation of the liquid aqueous mixture.

The aqueous mixture of fibrinogen component and fibrinogen activator may be mixed in any suitable container. According to certain preferred embodiments, the container used for mixing is a mold in which the liquid aqueous mixture is subsequently frozen. According to this embodiment, separate liquid aqueous solutions of the fibrinogen component and the fibrinogen activator are simultaneously introduced into the mold, thereby mixing the two solutions. Alternatively, separate liquid aqueous solutions of the fibrinogen component and the fibrinogen activator may be prepared in a container and then introduced into the mold.

The size and geometry of a given mold may be determined empirically by one skilled in the art depending on the desired size and shape of the solid dressing to be produced. Suitable materials for the mold include, but are not limited to, polymers such as polyvinyl chloride (PVC), glycol-modified polyethylene terephthalate, and polyethylene. Other suitable materials include metals such as stainless steel, paper, cardboard, and water-resistant paper or cardboard. The mold may also be made of a rapidly dissolving material that is solid at the temperature at which the mold is stored, and/or a material that can be lyophilized to a solid state.

According to the method of the present invention, forming a liquid aqueous mixture of the fibrinogen component and the fibrinogen activator is performed at a temperature sufficiently low to inhibit activation of the fibrinogen component by the fibrinogen activator.

Activation of the fibrinogen component by the fibrinogen activator can be determined by any suitable method known or available to those skilled in the art. For example, fibrin formation from activation of the fibrinogen component may be visually detected by observing deviations from a uniform surface condition in the resulting solid dressing. Firm, smooth and shiny "sheets" on the surface of the solid dressing, like excessive curling of the edges, are also signs of fibrin. In addition, dressings with high amounts of fibrin often have a mottled color and a significantly heterogeneous appearance after hydration.

Preferably, activation of the fibrinogen component can be assessed by various biochemical assays, such as the method described in U.S. patent application publication No. US2006/0155234 a 1. According to this test, the conversion of fibrinogen gamma chains to cross-linked gamma-gamma dimers can be used as an indication that the fibrinogen component is activated by the fibrinogen activator (the proportion of gamma chains converted to gamma-gamma dimers is related to the amount of activated fibrinogen).

Other biochemical assays can evaluate changes in the chains of other components of fibrinogen, such as conversion of the a α chain to free α chain and fibrinopeptide a, or conversion of the B β chain to free β chain and fibrinopeptide B. These changes can be monitored by gel electrophoresis in a similar manner as the conversion of gamma to gamma as described in U.S. patent application publication US 2006/0155234A 1.

Preferred liquid aqueous mixtures prepared using the method of the invention are generally free of detectable gamma-gamma dimers, although in some cases it may be acceptable for the dressing to contain up to 9% gamma-gamma dimer, even up to 38% gamma-gamma dimer. Similarly, preferred liquid aqueous mixtures may contain up to 60% free alpha chains and still function satisfactorily. Particularly preferred aqueous mixtures comprise less than 60% free alpha chains, more preferably less than 50%, even more preferably less than 40%, less than 30%, less than 20% or less than 10% free alpha chains. According to certain preferred embodiments, these values do not change significantly over time while the liquid aqueous mixture is maintained at a suitable temperature (preferably at 4 ℃ ± 2 ℃ or below 4 ℃ ± 2 ℃).

The temperature at which the liquid aqueous mixture is prepared is sufficient to inhibit activation of the fibrinogen component by the fibrinogen activator. Preferably, this temperature is from 2 ℃ to 8 ℃ or less than 2 ℃ to 8 ℃, more preferably 4 ℃ ± 2 ℃. Any suitable method may be used to achieve the desired temperature for preparing the liquid aqueous mixture.

According to certain preferred embodiments, the container or mold is cooled to the desired temperature by placing it in an environment having a temperature equal to or lower than the desired temperature. Preferably, the container or mould is cooled to a temperature substantially below the temperature required to prepare the liquid aqueous mixture. According to a particularly preferred embodiment, the container or mold is cooled by placing it in an environment having a temperature of about-80 ℃ for a sufficient time.

According to other embodiments, the aqueous solution of the fibrinogen component and the aqueous solution of the fibrinogen activator are cooled to a desired temperature prior to preparing the liquid aqueous mixture. This cooling may be achieved by any suitable method, such as placing a container containing the aqueous solution on ice.

After the liquid aqueous mixture has been prepared, it can be stored at a suitable temperature or it can be used directly to prepare the frozen aqueous mixture of the invention. Preferably, the frozen aqueous mixture is prepared directly using the liquid aqueous mixture.

According to certain preferred embodiments of the invention, the liquid aqueous mixture is then cooled to a temperature at which it becomes a frozen aqueous mixture of the fibrinogen component and the fibrinogen activator. This frozen aqueous mixture can be used directly or it can be stored at a suitable temperature.

Any method and technique known and available to those skilled in the art can be used to cool the liquid aqueous mixture to the desired temperature. For example, the liquid aqueous mixture may be introduced into a second vessel or mold that has been cooled to a temperature sufficient to cause the liquid aqueous mixture to freeze. Alternatively, the liquid aqueous mixture in the container or mold may be placed in an environment having a temperature sufficient to cause the liquid aqueous mixture to freeze. Such an environment may include, for example, a cooler set to a predetermined temperature, such as-5 ℃, 10 ℃, -15 ℃, -20 ℃, -25 ℃, -30 ℃, -40 ℃, -50 ℃, or-80 ℃. The mold may be arranged so that one or more surfaces thereof are in intimate contact with a surface that has been and/or is being cooled to a desired temperature. Alternatively, the mold or container containing the liquid aqueous solution may be placed directly on dry ice (-78 ℃), or in a suitable cold water bath, such as dry ice/acetone, dry ice/liquid nitrogen, or liquid nitrogen alone. The mold or vessel may also be placed in a stream of nitrogen gas produced by the vaporization of liquid nitrogen or other suitable cryogenic gaseous coolant stream. In such a case, it may be desirable for the gaseous stream to contact a single side of the mold to be cooled. In a more preferred embodiment, such streams may be directed onto two or more sides of the object to be cooled.

Preferred frozen liquid aqueous mixtures prepared using the method of the present invention are generally free of detectable gamma-gamma dimers, although in some cases it may be acceptable for the mixture to contain up to 9% gamma-gamma dimer, even up to 38% gamma-gamma dimer. Similarly, preferred frozen aqueous mixtures may contain up to 57% free alpha chains and still function satisfactorily. Particularly preferred frozen aqueous mixtures comprise less than 57% free alpha chains, more preferably less than 46%, even more preferably less than 46%, less than 31%, less than 20%, less than 16% or less than 10% free alpha chains. According to certain preferred embodiments, these values do not change significantly over time.

The frozen aqueous mixture can be stored or used directly for preparing solid dressings or for treating wounded tissue. Certain embodiments of the present invention relate to these frozen aqueous mixtures and to their use for the preparation of solid dressings or for the treatment of wounded tissue.

Preferably, when used to prepare a solid dressing, the frozen aqueous mixture is subjected to a process such as lyophilization or freeze-drying to reduce the moisture content to a desired level, i.e., to a level where the dressing is solid rather than frozen, and thus does not change shape or form significantly when standing upright, facing down the surface of the wound, at room temperature for 24 hours. Similar methods to achieve the same result, such as drying, spray drying, vacuum drying and vitrification, can also be used.

As used herein, "moisture content" refers to the amount of moisture freely available in the dressing. By "freely available" is meant that the water is not bound or complexed to one or more non-liquid components of the dressing. Moisture content as referred to herein refers to levels determined by methods substantially similar to the FDA approved modified Karl Fischer method (Meyer and Boyd, Analytical chem., 31: 215-219, 1959; May et al, J.biol.Standaryzation, 10: 249-259, 1982; Centers for biological Evaluation and research, FDA, Docket No.89D-0140, 83-93; 1990) or by near infrared spectroscopy.

Those skilled in the art can empirically determine the appropriate conditions for lyophilization or freeze-drying of the frozen aqueous mixture to form a solid dressing, including, but not limited to, its temperature and duration. Such conditions depend, for example, on the moisture content of the frozen aqueous mixture, the desired moisture content of the solid dressing, the environment in which the process is carried out, and the equipment used.

Solid dressings produced by the method of the present invention are also a preferred embodiment of the present invention. Such dressings contain sufficient fibrinogen component and sufficient fibrinogen activator to form a fibrin clot with or without additional hydration when applied to wounded tissue. Such dressings do not contain undesirable amounts of fibrin, whether or not they are cross-linked. One skilled in the art can empirically determine whether a particular amount of fibrin is undesirable based on: predetermined properties of the solid dressing such as appearance quality, flexibility, hydration time, etc.; and its intended use (e.g., a dressing designed for exuding wounds can tolerate a greater amount of fibrin than a dressing designed for arterial wounds).

The amount of fibrin present in a given dressing may be determined by any suitable method known and available to those skilled in the art. For example, deviations from a homogeneous surface appearance can be observed to visually detect the amount of fibrin in a particular dressing. A strong, smooth and shiny "sheet" on the surface of the solid dressing, like an excessive curling of the edges, is also an indication of fibrin. In addition, dressings with high amounts of fibrin often have a mottled color and a significantly heterogeneous appearance after hydration.

Alternatively, the amount of fibrin may also be evaluated by various biochemical tests, such as the method described in U.S. patent application publication US2006/0155234 a 1. According to this test, the conversion of fibrinogen gamma chains to cross-linked gamma-gamma dimers serves as an indication of the presence of fibrin (the proportion of gamma chains converted to gamma-gamma dimers is a measure of the amount of fibrin produced).

Preferred solid dressings prepared using the method of the present invention are generally free of detectable gamma-gamma dimer, although in some cases it may be acceptable for the mixture to contain up to 9% gamma-gamma dimer, and even up to 38% gamma-gamma dimer. Similarly, preferred frozen aqueous mixtures may contain up to 57% free alpha chains and still function satisfactorily. Particularly preferred frozen aqueous mixtures comprise less than 57% free alpha chains, more preferably less than 46%, even more preferably less than 46%, less than 31%, less than 20%, less than 16% or less than 1O% free alpha chains.

According to certain preferred embodiments, the haemostatic layer of the solid dressing may also comprise a binder in order to promote or improve adhesion between layers and/or layers to any supporting layer. Illustrative examples of suitable binders include, but are not limited to, sucrose, mannitol, sorbitol, gelatin, hyaluronidase and its derivatives such as hyaluronic acid, maltose, povidone, starch, chitosan and its derivatives, and cellulose derivatives such as carboxymethylcellulose, and mixtures of two or more thereof. Such a binder may be introduced into an aqueous mixture of the fibrinogen component and the fibrinogen activator, or may be introduced into an aqueous solution of the fibrinogen component and/or an aqueous solution of the fibrinogen activator prior to mixing.

The haemostatic layer of the solid dressing may also optionally contain one or more suitable fillers such as sucrose, lactose, maltose, silk, fibrin, collagen, albumin, polysorbate (Tween), chitin, chitosan and its derivatives such as NOCC-chitosan, alginic acid and its salts, cellulose and its derivatives, proteoglycans, hyaluronidase and its derivatives such as hyaluronic acid, glycolic acid polymers, lactic acid polymers, glycolic acid/lactic acid copolymers, and mixtures of two or more thereof. Such fillers may be introduced into the aqueous mixture of the fibrinogen component and the fibrinogen activator, or may be introduced into the aqueous solution of the fibrinogen component and/or the aqueous solution of the fibrinogen activator prior to mixing.

The haemostatic layer of the solid dressing may also optionally contain one or more suitable solubilising agents such as sucrose, dextrose, mannose, trehalose, mannitol, sorbitol, albumin, hyaluronidase and derivatives thereof such as hyaluronic acid, sorbate, polysorbate (Tween), Sorbitan (SPAN), and mixtures of two or more thereof. Such a solubilising agent may be introduced into the aqueous mixture of the fibrinogen component and the fibrinogen activator, or may be introduced into the aqueous solution of the fibrinogen component and/or the aqueous solution of the fibrinogen activator prior to mixing.

The haemostatic layer of the solid dressing may also optionally comprise one or more suitable foaming agents, such as a mixture of a physiologically acceptable acid (e.g. citric acid or acetic acid) and a physiologically suitable base (e.g. sodium bicarbonate or calcium carbonate). Other suitable foaming agents include, but are not limited to, dry particles containing a pressurized gas, such as sugar particles containing carbon dioxide (see, e.g., U.S. patent 3,012,893) or other physiologically acceptable gases (e.g., nitrogen or argon), and pharmacologically acceptable peroxides. Such foaming agents may be introduced into the aqueous mixture of the fibrinogen component and the fibrinogen activator, or may be introduced into the aqueous solution of the fibrinogen component and/or the aqueous solution of the fibrinogen activator prior to mixing.

The haemostatic layer of the solid dressing may also optionally contain a suitable source of calcium ions, for example calcium chloride, and/or a fibrin cross-linking agent such as a transaminase (e.g. factor VIII/XIIIa) or glutaraldehyde. Such a source of calcium ions and/or a fibrin crosslinking agent may be introduced into the aqueous mixture of the fibrinogen component and the fibrinogen activator, or may be introduced into the aqueous solution of the fibrinogen component and/or the aqueous solution of the fibrinogen activator prior to mixing.

The appropriate moisture content for a particular solid dressing may be determined empirically by one skilled in the art based on its intended application.

For example, in certain embodiments of the present invention, higher moisture content is associated with softer solid dressings. Thus, in solid dressings designed for wounds of the extremities, a moisture content of at least 6%, and even in the range of 6% -44% is preferred.

Likewise, in other embodiments of the invention, lower moisture content is associated with more rigid solid dressings. Thus, in solid dressings designed for smoothing wounds, such as abdominal or thoracic wounds, a moisture content of less than 6% is preferred, even more preferably in the range of 1% -6%.

Illustrative examples of suitable moisture content of the solid dressing therefore include, but are not limited to, the following (each value being ± 0.9%): less than 53%, less than 44%, less than 28%, less than 24%, less than 16%, less than 12%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2.5%, less than 2%, less than 1.4%, 0-12% (not inclusive), 0-6%, 0-4%, 0-3%, 0-2%, 0-1%, 1-16%, 1-11%, 1-8%, 1-6%, 1-4%, 1-3%, 1-2%, and 2-4%.

The fibrinogen component of the haemostatic layer of the solid dressing may be any suitable fibrinogen known and available to those skilled in the art. The fibrinogen component may also be a functional derivative or metabolite of fibrinogen, such as fibrinogen alpha, beta and/or gamma chains, soluble fibrin I or fibrin II, or a mixture of two or more thereof. The particular fibrinogen (or functional derivative or metabolite) for a particular application may be selected empirically by one skilled in the art. As used herein, the term "fibrinogen" is meant to include fibrinogen and small amounts of factor XIII/factor XIIIa, or some other such transaminase mixture. Such minor amounts are generally considered by those skilled in the art to be the amounts found in mammalian fibrinogen after purification according to methods and techniques currently known and available in the art, and may be any amount, typically 0.1-20 units/mL.

Preferably, the fibrinogen used as the fibrinogen component of the solid dressing is purified fibrinogen suitable for introduction into a mammal. Typically, this fibrinogen is part of a mixture of human plasma proteins comprising factors XIII/XIIIa which have been purified to appropriate levels and virus inactivated. The preferred aqueous fibrinogen solution used to prepare the solid dressing contains about 37.5mg/mL of fibrinogen and has a pH of about 7.4 ± 0.1, although a pH range of 5.5-8.5 is acceptable. Suitable fibrinogen for use as the fibrinogen component has been described in the art, for example, U.S. patent 5,716,645, and similar materials are commercially available, for example, from Sigma-Aldrich, Enzyme Research Laboratories, Haematologic Technologies, and Anira.

Preferably, the fibrinogen component is present in an amount of about 1.5 to about 13.0mg (+ -0.9 mg) fibrinogen per square centimeter of solid dressing, more preferably about 3.0 to about 13.0mg/cm². However, larger or smaller amounts may be used depending on the particular application of the solid dressing. For example, according to certain embodiments wherein increased adherence is desired, the fibrinogen component is present in an amount of about 11.0 to about 13.0mg (± 0.9mg) per square centimeter of solid dressing. Likewise, lower levels of fibrinogen component may be used according to certain embodiments designed to treat vessels containing low pressure.

The fibrinogen activator used in the haemostatic layer of the solid dressing may be any substance or mixture of substances known to those skilled in the art to convert fibrinogen to fibrin. Illustrative examples of suitable fibrinogen activators include, but are not limited to, the following: thrombin, such as human thrombin or bovine thrombin, and prothrombin, such as human prothrombin or prothrombin complex concentrate (mixture of coagulation factors II, VII, IX and X); snake venoms, such as batroxobin, reptilian thrombin (a mixture of batroxobin and factor XIIIa), brazilian spearhead snake enzyme (bothrombin), snake venom thrombin (calobin), fibrizyme, and enzymes isolated from venom of Bothrops jararacusssus; and mixtures of any two or more of these. See, e.g., Dascombe et al, Thromb. Haemost.78: 947-51 (1997); hahn et al, j. biochem. (Tokyo) 119: 835-43 (1996); fortova et al, j.chromatogr.s.biomed.appl.694: 49-53 (1997); and Andriao-Escarso et al, toxicon.35: 1043-52(1997).

Preferably, the fibrinogen activator is thrombin. More preferably, the fibrinogen activator is mammalian thrombin, although bird and/or fish thrombin may also be used where appropriate. While any suitable mammalian thrombin may be used in the solid dressing, for the intended use of the solid dressing, the thrombin used in the haemostatic layer is preferably a lyophilized mixture of human plasma proteins which have been substantially purified and virally inactivated. Suitable thrombin may be obtained from commercial sources such as Sigma-Aldrich, Enzyme research laboratories, Haematologic Technologies and BiomolInternational. A particularly preferred aqueous thrombin solution for preparing the solid dressing comprises thrombin having a potency of about 10-2000. + -.50 International units/mL, more preferably a potency of about 25. + -.2.5 International units/mL. Other components may include albumin (typically about 0.1mg/mL) and glycine (typically about 100mM + -0.1 mM). The pH of this particularly preferred aqueous thrombin solution is generally in the range of 6.5 to 7.8, preferably 7.4. + -. 0.1, although a pH range of 5.5 to 8.5 is acceptable.

In addition to the haemostatic layer, the solid dressing may optionally further comprise one or more support layers. As used herein, a "support layer" refers to a material that maintains or improves the structural integrity of a solid dressing and/or maintains or improves the structural integrity of a fibrin clot formed when such a dressing is applied to wounded tissue.

According to certain preferred embodiments of the present invention, the support layer comprises a padding material on a side opposite to the side applied to the wounded tissue. Such liner materials may be attached by a physiologically acceptable adhesive, or may be self-adhering (e.g., by having sufficient surface static charge). Preferably, the backing material is placed in a mould or container prior to introducing the liquid aqueous mixture or solution of the fibrinogen component and the fibrinogen activator. The physiologically acceptable adhesive may be placed on the backing material prior to introducing the liquid aqueous mixture or solution of the fibrinogen component and the fibrinogen activator.

The liner material may comprise one or more resorbable materials or one or more non-resorbable materials or a mixture thereof. Preferably, the padding material is a purely resorbable material.

Any suitable resorbable material known and available to those skilled in the art may be used in the present invention. For example, the resorbable material may be a proteinaceous substance, such as silk, fibrin, keratin, collagen, and/or gelatin. Alternatively, the resorbable material may be a carbohydrate substance such as alginate, chitin, cellulose, proteoglycans (e.g., poly-N-acetylglucosamine), hyaluronidase and its derivatives (e.g., hyaluronic acid), glycolic acid polymers, lactic acid polymers, or glycolic acid/lactic acid copolymers. The resorbable material may also comprise a mixture of proteinaceous substances or a mixture of carbohydrate substances or a mixture of proteinaceous substances and carbohydrate substances. The particular resorbable material may be empirically selected by one skilled in the art based on the intended application of the solid dressing.

According to certain preferred embodiments of the present invention, the resorbable material is a carbohydrate substance. Illustrative examples of particularly preferred resorbable materials include, but are not limited to, the materials sold under the trade names Vicryl (a glycolic acid/lactic acid copolymer) and DEXON (a glycolic acid polymer).

Any suitable non-resorbable material known and available to those skilled in the art may be used as the padding material. Illustrative examples of suitable non-resorbable materials include, but are not limited to, plastics, silicone polymers, paper and paper products, latex, gauze, and the like.

According to other preferred embodiments, the support layer comprises an internal support material. Such an inner support material is preferably completely contained within the haemostatic layer of the solid dressing, although in certain embodiments it may be located between two adjacent haemostatic layers. Preferably, the internal support material is placed in the mold or container during introduction of the liquid aqueous mixture or solution of the fibrinogen component and the fibrinogen activator.

As in the case of the liner material, the inner support material may be a resorbable or non-resorbable material, or a mixture thereof, such as a mixture of two or more resorbable materials or a mixture of two or more non-resorbable materials or a mixture of resorbable and non-resorbable materials.

According to other preferred embodiments, the support layer may comprise a frontal support material on the wound-facing side of the dressing (i.e. the side to be applied to the wounded tissue). As in the case of the cushion material and the inner support material, the front support material may be a resorbable or non-resorbable material, or a mixture thereof, e.g. a mixture of two or more resorbable materials or a mixture of two or more non-resorbable materials may be a mixture of a resorbable and non-resorbable material.

According to other preferred embodiments, the solid dressing comprises both a backing material and an inner support material in addition to the haemostatic layer, that is, the solid dressing comprises two support layers in addition to the haemostatic layer. According to other preferred embodiments, the solid dressing comprises both a frontal support material and an internal support material in addition to the haemostatic layer. According to other preferred embodiments, the solid dressing comprises a backing material in addition to the haemostatic layer, a front support material and an inner support material.

The various layers of the dressing of the invention may be attached to each other by any suitable method known and available to those skilled in the art. For example, a physiologically acceptable sealant can be applied to the liner material (when present) and a layer of hemostatic agent subsequently attached thereto.

In certain embodiments of the invention, the shear strength and/or structure of the physiologically acceptable sealant is such that the backing material can be separated from the fibrin clot formed by the haemostatic layer after the dressing is applied to the wounded tissue. In other embodiments, the shear strength and/or structure of the physiologically acceptable sealant is such that the backing material can be separated from the fibrin clot formed by the haemostatic layer after the dressing is applied to the wounded tissue.

Suitable fibrinogen and suitable fibrinogen activators for the haemostatic layer of the solid dressing may be obtained from any suitable source known and available to those skilled in the art, including but not limited to the following: from commercial suppliers such as Sigma-Aldrich and Enzyme Research Laboratories; extraction and purification from human or mammalian plasma by any method known and available to those skilled in the art; supernatant or paste derived from plasma or recombinant tissue cultures, viruses, yeasts, bacteria, etc., containing genes introduced according to standard recombinant DNA techniques expressing human or mammalian plasma proteins; and/or from a bodily fluid (e.g., blood, milk, lymph, urine, etc.) of a transgenic mammal (e.g., goat, sheep, cow) that contains genes that have been introduced according to standard transgenic techniques and that express the desired fibrinogen and/or the desired fibrinogen activator.

According to certain preferred embodiments of the invention, the fibrinogen is mammalian fibrinogen, such as bovine fibrinogen, porcine fibrinogen, ovine fibrinogen, equine fibrinogen, caprine fibrinogen, feline fibrinogen, canine fibrinogen, murine fibrinogen or human fibrinogen. According to other embodiments, the fibrinogen is bird fibrinogen or fish fibrinogen. According to any of these embodiments, the fibrinogen may be recombinantly produced fibrinogen or transgenic fibrinogen.

According to certain preferred embodiments of the invention, the fibrinogen activator is a mammalian thrombin, e.g., bovine thrombin, porcine thrombin, ovine thrombin, equine thrombin, caprine thrombin, feline thrombin, canine thrombin, murine thrombin and human thrombin. According to other embodiments, the thrombin is avian thrombin or fish thrombin. According to any of these embodiments, the thrombin may be recombinantly produced thrombin or transgenic thrombin.

As a general proposition, the purity of the fibrinogen and/or fibrinogen activator used in the solid dressing should be that known by those skilled in the relevant art to produce the best potency and stability of the protein. Preferably, the fibrinogen and/or fibrinogen activator has been subjected to multiple purification steps, such as precipitation, concentration, diafiltration and affinity chromatography (preferably immunoaffinity chromatography), in order to remove substances that cause fragmentation, activation and/or degradation of the fibrinogen and/or fibrinogen activator during production, storage and/or use of the solid dressing. Illustrative examples of such substances that are preferably removed by purification include: protein contaminants such as meta-alpha trypsin inhibitors and pre-alpha trypsin inhibitors; non-protein contaminants, such as lipids; and mixtures of proteinaceous and non-proteinaceous contaminants, such as lipoproteins.

The amount of fibrinogen activator used in the solid dressing is preferably selected to optimize its efficacy and stability. Likewise, suitable concentrations for a particular application of the solid dressing may be determined empirically by one skilled in the relevant art. According to certain preferred embodiments of the invention, when the fibrinogen activator is human thrombin, the amount of human thrombin employed is between 2.50 units/mg of the fibrinogen component and 0.025 units/mg of fibrinogen (all values are ± 0.001). Other preferred embodiments relate to similar solid dressings in which the amount of thrombin is from 0.250 units/mg fibrinogen to 0.062 units/mg fibrinogen and to solid dressings in which the amount of thrombin is from 0.125 units/mg fibrinogen to 0.080 units/mg fibrinogen.

In using a solid dressing, it is preferred that the fibrinogen and fibrinogen activator are activated by endogenous fluid of the patient flowing from the bleeding wound when the dressing is applied to wounded tissue. Alternatively, in situations where the fluid flow from the wounded tissue is insufficient to provide adequate hydration of the protein layer, the fibrinogen component and/or thrombin may be activated by a suitable, physiologically acceptable liquid, optionally containing any necessary cofactors and/or enzymes, prior to or during application of the dressing to the wounded tissue.

In some embodiments of the invention, the haemostatic layer may also contain one or more supplements, such as growth factors, drugs, polyclonal and monoclonal antibodies and other compounds. Illustrative examples of such supplements include, but are not limited to, the following: fibrinolysis inhibitors such as aprotinin (aprotonin), tranexamic acid, and epsilon-aminocaproic acid; antibiotics, such as tetracycline and ciprofloxacin, amoxicillin, and metronidazole; anticoagulants such as activated protein C, heparin, prostacyclin, prostaglandins (in particular (PGI2), leukotrienes, antithrombin III, ADPase, and plasminogen activators; steroids such as dexamethasone and inhibitors of prostacyclin, prostaglandins, leukotrienes and/or cytokinins to inhibit inflammation; cardiovascular drugs such as calcium channel blockers, vasodilators and vasoconstrictors; chemoattractants; local anesthetics such as bupivacaine; and antiproliferatives/antineoplastics such as 5-fluorouracil (5-FU), taxol and/or taxotere; antivirals such as gancyclovir (ganciclovir), zidovudine, amantadine, vidarabine, ribavirin (ribavain), trifluridine, acyclovir, dideoxyuridine, and antibodies to viral components or gene products; cytokines, such as alpha-or beta-or gamma-interferon, alpha-or beta-tumor necrosis factor, and interleukins; a colony stimulating factor; erythropoietin; antifungal agents, such as, for example, hibitane, ketoconazole, and nystatin; parasiticides, such as pentamidine; anti-inflammatory agents, such as alpha-1-anti-trypsin and alpha-1-anticlotting lactase; anesthetics, such as bupivacaine; an analgesic; a preservative; a hormone; vitamins and other nutritional supplements; a glycoprotein; (ii) an adhesion protein; peptides and proteins; carbohydrates (simple and/or complex carbohydrates); a proteoglycan; anti-angiogenin; an antigen; a lipid or liposome; oligonucleotides (sense and/or antisense DNA and/or RNA); and gene therapy agents. In other embodiments of the invention, the backing layer and/or the inner support layer, if present, may contain one or more supplements. According to certain preferred embodiments of the present invention, the therapeutic supplement is present in an amount greater than its solubility limit in fibrin.

The following examples are illustrative only and are not intended to limit the scope of the invention as defined by the claims which follow. It will be apparent to those skilled in the art that various modifications and variations can be made in the method of the present invention without departing from the spirit and scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the following claims and their equivalents.

Detailed description of the preferred embodiments

Examples

The ability of the dressing to seal damaged vessels was determined by an Ex Vivo Porcine Arteriotomy (EVPA) performance test, which was first described in us patent 6,762,336. The EVPA performance test evaluates the ability of the dressing to prevent fluid flow through the pores in the porcine artery. Although the method described in us patent 6,762,336 has proven useful for evaluating hemostatic dressings, it does not accurately replicate the conditions required for in vivo success. More specifically, the method disclosed in us patent 6,762,336 requires testing at 37 ℃, whereas in practical situations the wound temperature is typically below this temperature. This reduced temperature can significantly reduce the rate of fibrin formation and its hemostatic efficacy in trauma patients. See, e.g., Acheson et al, j. trauma 59: 865-874(2005). The test in us 6,762,336 has not required a high degree of adhesion of the dressing to the damaged tissue. Thus, the failure mode in which fibrin is formed but the dressing fails to adhere tightly to the tissue cannot be detected by this test. In addition, in the case of treating certain trauma patients, the pressure used in the method (200mHg) may be exceeded. Overall, as a result, numerous animal trials typically involving small animals (e.g., rats and rabbits) must be conducted in order to correctly predict dressing performance in large animals, realistic wound studies, and in a clinical setting.

To minimize the amount of time required to develop the invention and the number of animal studies, improved ex vivo testing methods were developed. To this end, the basic conditions under which the dressing tests were performed were varied and the severity of the test parameters was increased to include tests at lower temperatures (i.e. 29-33 ℃ vs. 37 ℃ C., representing actual physiological attacks of wound temperature (Acheson et al, J. Trauma 59: 865-.

In addition, new tests were developed to directly measure the adhesion of the dressing to the damaged tissue. Both of these tests show improved stringency and can therefore outperform previous ex vivo tests and can replace many in vivo tests in efficacy.

The following is a list of abbreviations used in the following examples: CFB: intact fibrinogen buffer (100mM sodium chloride, 1.1mM calcium chloride, 10mM Tris, 10mM sodium citrate, 1.5% sucrose, human serum albumin (80mg/g total protein) and Tween80 (animal derived) 15mg/g total protein) CTB: complete thrombin buffer (150mM sodium chloride, 40mM calcium chloride, 10mM Tris and 100mM L-lysine with the addition of 100. mu.g/ml HSA) ERL: enzyme Research laboratory evpa: ex vivo porcine arteriotomy FD: the hemostatic dressing HSA of the present invention: human serum albumin HD: the "sandwich" fibrin sealant hemostatic dressing IFB disclosed in us patent 6,762,336: incomplete fibrinogen buffer. (ii) a CFBPETG without HSA and Tween: ethylene glycol-modified polyethylene terephthalate PPG: polypropylene PVC: polyvinyl chloride TRIS: tris (2-amino-2-hydroxymethyl-1, 3-propanediol) example 1

The gasket material (DEXON) was cut and placed in a 2.4X2.4cm per PETG die. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes. Fibrinogen was formulated in CFB (Enzyme Research Laboratories). The final pH of fibrinogen was 7.4. + -. 0.1. Fibrinogen concentrations were adjusted to 37.5, 31.7, 25.9, 20.16, 14.4, 8.64, and 4.3 mg/ml. This would result in 13, 11, 9, 7, 5, 3 or 1.5mg/cm when 2ml of fibrinogen is delivered into the mould²Fibrinogen dosage of (a). After preparation, fibrinogen was placed on ice prior to use. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. The concentration of thrombin was adjusted so that when mixed with the fibrinogen solution as described below, the composition produced a solution containing 0.1 units/mg fibrinogen. After preparation, thrombin was placed on ice prior to use. The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on a copper plate, which was placed on top of dry ice. The repeat pipettor was filled with fibrinogen and the second repeat pipettor was filled with thrombin. 2ml of fibrinogen and 300 microliters of thrombin were simultaneously dispensed into each mold. After filling the molds with the reagents, they were frozen and then returned to the-80 ℃ cooler for at least 2 hours. The frozen dressing was then placed in precooled Genesis^TMIn a freeze-dryer (Virus, Gardiner, N.gamma.). The cell was sealed and the temperature was allowed to equilibrate. The chamber is then evacuated and the dressing is lyophilized through primary and secondary drying cycles.

The dressing was removed from the freeze-dryer, sealed in foil bags and stored at room temperature prior to testing. Subsequently, the dressing was evaluated for EVPA, adhesion and weight tests.

The results are given in the following table and graphically represented in fig. 3A-3C.

Group of	EVPA pass/total	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						13mg/cm²	6/6	4.0	0.0	198.0	12.6
11mg/cm²	6/6	3.8	0.4	163	48.5
						9mg/cm²	5/6	3.0	0.0	88	20.0
7mg/cm²	6/6	3.2	0.4	93	17.6
						7mg/cm²	5/6	3.0	0.0	94.7	8.2
5mg/cm²	5/5	2.8	0.4	76	34.2
						3mg/cm²	5/5	2.4	0.5	48	27.4
1.5mg/cm²	0/6	0.1	0.2	4.7	11.4

Example 2

Monolithic dressings were produced as follows: the liner material was cut and placed in each petg2.4x2.4cm die. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes.

For all dressings, ERL fibrinogen was formulated in CFB, lot 3114. The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin was adjusted to deliver 0.1 units/mg fibrinogen or 25 units/ml thrombin. After preparation, thrombin was placed on ice prior to use. The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on a copper plate precooled on the cooler. The repeat pipettor was filled with fibrinogen and the second repeat pipettor was filled with thrombin. 2ml of fibrinogen and 300 microliters of thrombin were simultaneously dispensed into each mold. After filling the molds with reagents, they were returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. The dressing was then lyophilized as described above. After completion, the dressing was stored in a low moisture transfer foil pouch containing 5 grams of desiccant.

Using the same materials as above, as described above¹Three layers of dressings were produced. The dressing was then left at 100% relative humidity and 37 ℃ for various periods of time to increase its relative moisture content to the desired level. The dressings were visually evaluated and their hand and other physical properties were evaluated. After this evaluation, samples of each dressing were tested to determine their moisture content. The remaining dressings were tested for performance in EVPA, adhesion and weight retention tests. Results

The results of the tests are given in the following table: table 1: performance data for the solid dressing of the invention

Exposure time to 100% humidity at 37 deg.C (minutes)	Water content%	EVPA # PASS/TOTAL	Peel test adhesion (+ -Standard deviation)	Weight maintenance (g) (mean. + -. standard deviation)
					0	2.5	2/2	4.0±0	148±28.3
1	5.8	2/2	3.5±0.71	123±7.1
					15	16	2/2	2.5±0.71	108±14.1
45	24	2/2	4.0±0	168±0
					60	28	2/2	4.0±0	273±7.1
225	44	2/2	2±0	58±14.1
					1200	52	ND	ND	ND

Table 2: performance data for triple layer dressings

Exposure time to 100% humidity at 37 deg.C (minutes)	Water content%	EVPA # PASS/TOTAL	Peel test adhesion	Weight maintenance (g) (average)
					0	3	1/1	2.0	78
15	22	1/1	2.0	78
					60	33.7	0/1	0.5	28

Table 3: integrity and feel characteristics of the solid dressings of the present inventionTable 4: integrity and feel characteristics of triple layer dressingsAnd (4) conclusion:

the monolithic dressing is capable of adequately functioning at very high moisture levels. It was found that almost 28% of the water remained fully functional. At elevated moisture levels up to 44%, the dressings were still functional, however some of their activity was reduced. Higher moisture levels may also retain some functionality. The original dressing was not soft at a moisture content of 2.5%, but had all other desirable properties after hydration, including appearance, flat surface, integrity, rapid and uncomplicated hydration, and a smooth appearance. Once the moisture content is increased to 5.8%, the monolithic dressing becomes flexible, but still retains its functionality and desirable characteristics. They retain their softness without curling or losing their integrity or appearing to form too much fibrin prior to hydration.

This is in contrast to tri-layer dressings, which begin to lose their desirable characteristics when water is added, and are completely lost when water is increased to 33%. Example 3

For dressings using a liner, the liner material was cut and placed in each petg2.4x2.4cm mold. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes. For the dressing without the backing material, the PETG2.4x2.4cm mold was placed in a-80 ℃ cooler for at least 60 minutes.

For all dressings, ERL fibrinogen was formulated in CFB, lot 3114. The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin was adjusted to deliver 0.1 units/mg fibrinogen or 25 units/ml thrombin. After preparation, thrombin was placed on ice prior to use. The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on a copper plate, which was placed on top of dry ice. The repeat pipettor was filled with fibrinogen and the second repeat pipettor was filled with thrombin. 2ml of fibrinogen and 300 microliters of thrombin were simultaneously dispensed into each mold. After filling the molds with reagents, they were returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. The dressing was then lyophilized as described below.

Performance tests were performed on both groups in the EVPA test. In addition, the group with the liner was also tested in adhesion and weight retention tests. As a result:

group of	EVPA pass/total	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						Liner pad	6/6	3.7	0.5	153	37.3
Linerless gasket	9/12

And (4) conclusion:

dressings formulated with backing materials worked well, all passed the EVPA test, and had high adhesion and weight retention values. Dressings without backing material were less effective in the EVPA test, however surprisingly 75% of them passed the EVPA test. No liner was present and no further tests were performed. Dressings made without a liner have been successfully tested by EVPA and have been shown to be effective in treating arterial lesions and even more effective in treating venous and small vessel lesions. Example 4

For all dressings, ERL fibrinogen, lot 3130 was formulated in CFB. The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin was adjusted to deliver 0.1 units/mg fibrinogen or 25 units/ml thrombin. For the group with the shredded Vicryl mesh dispersed inside, this support material was cut into pieces of about 1mmx1mm and dispersed in a thrombin solution before filling the mold. After preparation, thrombin was placed on ice prior to use. The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. A cylindrical mold made of a 10mL or 3mL polypropylene syringe (Becton Dickinson) with the Luer lock cuff terminating end removed was used. The piston was withdrawn to 6mL and 2mL marks, respectively. For dressings utilizing pads, the support material is cut and placed in each mold and pressed downward until it abuts the piston. After preparation, the mold was placed upright and surrounded with dry ice, leaving an exposed opening at the top. 1mL of fibrinogen and 0.15mL of thrombin (with or without liner material dispersed inside) were dispensed into a 10mL mold, and 1mL of fibrinogen and 0.15mL of thrombin (with or without liner material dispersed inside) were dispensed into a 3mL mold and allowed to freeze for 5 minutes. The molds were then placed in a-80 ℃ cooler for at least 2 hours before being placed in a freeze-dryer and lyophilized as described above.

After removal from the freeze-dryer, both groups were performance tested in a modified EVPA test. In short, the plastic foam model slides over the artery. This cover has a hole therein corresponding to the hole in the artery and surrounding tissue. Warm saline was added to the surface of the dressing and the mold was immediately transferred through the holes in the foam to the arterial surface. The plunger was then depressed and held by hand for 3 minutes, after which the mold was withdrawn and the plunger was continued to be pressed. At this point, the artery was pressurized and the test continued as previously described. Results

Support material	Size of die	EVPA results at 250mmHg	Maximum pressure
				Is free of	10ml	By passing	＞250mmHg
Dexon Mesh Backing	10ml	By passing	Same as above
				Same as above	3ml	By passing	Same as above
Shredded Dexon mesh (discrete)	10ml	By passing	Same as above
				Same as above	3ml	Failure of	150mm Hg

And (4) conclusion:

dressings that did not include a pad or a DEXON mesh pad worked well, all passing the EVPA test at 250 mmHg. The dressing also worked well when the support material was dispersed throughout the composition, with the large size (10mL mold) dressing maintaining an intact 250mmHg pressure, while the smaller dressing maintained a pressure of 150 mmHg. This indicates that the use of a support material is optional and its location may be on the 'backing' of the dressing as desired, or dispersed throughout the composition. Example 5

A dressing with a support material on the "back" of the dressing (i.e., the side of the wound not facing the wound) was produced as follows: the web support material was first cut and placed in each PETG 10X10cm mold. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes.

For dressings with support material on the "front side" (i.e. the side facing the wound) of the dressing, these dressings are produced without any support material in the mould. The support mesh was placed on top of the dressing immediately after dispensing the fibrinogen and thrombin into the mould (see below) and gently pressed into the surface before it freezes. In all other aspects, the production of the dressing is as follows.

For all dressings, ERL fibrinogen was formulated in CFB, lot 3114. The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin was adjusted to deliver 0.1 units/mg fibrinogen or 25 units/ml thrombin. After preparation, thrombin was placed on ice prior to use. Thrombin was adjusted to deliver 0.1 units/mg fibrinogen or 25 units/ml thrombin. After preparation, thrombin was placed on ice prior to use. The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate placed on dry ice. The aluminum plate had a 0.25 inch hole drilled in the middle and a fitting was attached so that a length of tubing could be connected to a vacuum source. The mold was centered over the hole in the aluminum plate and the vacuum was turned on. Vacuum has two functions: preventing the mold from moving and keeping it flat with respect to the aluminum plate. 35 ml of fibrinogen and 5.25 ml of thrombin were placed in a 50ml tube, inverted three times and poured into a mold. After filling the molds and applying the support material as described above, they were returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. The dressing was then lyophilized as previously described.

orientation of support material (web)	EVPA # PASS/TOTAL	Adhesion test score	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						Back (far from injured position)	6/6	3.5	0.5	136	49
Front side (close to the injured position)	6/6	3.8	0.4	163	32

And (4) conclusion:

dressings formulated with any orientation of backing material worked well, all passed the EVPA test, and had high adhesion and weight retention values. This means that the support material may optionally be on the 'back' side of the dressing, or on the 'front' side of the composition. Example 6

The liner material (DEXON) was placed in a PETG2.4X2.4cm mold. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes.

Fibrinogen was formulated in CFB (Enzyme research laboratories (ERL), lot number 3114). The fibrinogen concentration was adjusted to 37.5mg/ml using CFB. The final pH of fibrinogen was 7.4. + -. 0.1. After preparation, fibrinogen was placed on ice prior to use.

Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. The thrombin concentration was adjusted with CTB to produce 12.5 units/mg fibrinogen (when mixed) which corresponded to 3120 units/ml thrombin prior to mixing. After preparation, thrombin was placed on ice prior to use.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on a copper plate that was placed on dry ice with a pre-cooling. The repeat pipettor was filled with fibrinogen and the second repeat pipettor was filled with thrombin. 2ml of fibrinogen and 300 microliters of thrombin were simultaneously dispensed into each mold. After filling the molds with reagents, they were returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. They were then lyophilized as described below and tested for performance using the EVPA and adhesion tests as described below. These results are shown in fig. 4A and 4B. Example 7

The gasket material was placed in each 1.5X1.5cm PVC mold. 15 microliters of 2% sucrose was pipetted onto the top of each of the four corners of the liner material. A second sheet of PETG plastic was fitted on top of the 1.5X1.5 mold and fixed in place. This forms a closed mold. The mold was then placed in a-80 ℃ cooler for at least 60 minutes. Fibrinogen was formulated in CFB (ERL, batch 3100). The fibrinogen concentration was adjusted to 37.5mg/ml using CFB. The final pH of fibrinogen was 7.4. + -. 0.1. After preparation, fibrinogen was placed on ice prior to use. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. CTB was used to modulate thrombin concentration so as to deliver the following amounts: 2.5, 0.25, 0.1, 0.05, 0.025, 0.016, 0.0125 and 0.01 units/mg of fibrinogen (when mixed) which corresponds to 624, 62.4, 25, 12.5, 6.24, 3.99, 3.12 and 2.5 units/ml of thrombin prior to mixing. After preparation, thrombin was placed on ice prior to use. The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was then removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. A 18 gauge needle was used to punch 3 holes in the top of the mold. One hole was used for injection of fibrinogen, a second for injection of thrombin and a third hole was used as a vent to release air displaced from the interior of the mold. The pipettor was then filled with fibrinogen and the second pipettor was filled with thrombin. 0.78ml of fibrinogen and 0.17ml of thrombin were simultaneously injected into each mold by these pipettors. After filling, the mold was placed on top of a liquid nitrogen bath for 30 seconds, then returned to a-80 ℃ cooler for at least 2 hours before being placed in a freeze-dryer. They were then lyophilized as described below and tested for performance using the EVPA and adhesion tests as described below. These results are shown in fig. 4A and 4B. Example 8

The liner material was placed in a 2.4X2.4cm PVC mold. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes. Fibrinogen was formulated in CFB (ERL, batch 3100). The fibrinogen concentration was adjusted to 37.5mg/ml using CTB. The final pH of fibrinogen was 7.4. + -. 0.1. After preparation, fibrinogen was placed on ice prior to use. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. CTB was used to modulate thrombin concentration so as to deliver the following amounts: 0.125, 0.025, 0.0125, 0.00625 and 0.0031 units/mg of fibrinogen (when mixed) which corresponds to 31.2, 6.24, 3.12, 1.56 and 0.78 units/ml of thrombin prior to mixing. After preparation, thrombin was placed on ice prior to use. The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. A 3ml syringe fitted with an 18 gauge needle was filled with 2ml of fibrinogen and a 1ml second syringe fitted with a22 gauge needle was filled with 0.3ml of thrombin. The contents of both syringes were dispensed simultaneously into each mold. After filling, the mold was placed on top of the liquid nitrogen for 30 seconds, then returned to the-80 ℃ cooler for at least 2 hours before being placed in the freeze-dryer. They were then lyophilized as described below and tested for performance using the EVPA and adhesion tests as described below. These results are shown in fig. 4A and 4B. Example 9

The liner material was placed in a 2.4X2.4cm PVC mold. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes. The vial containing 3 grams of fibrinogen (SigmaLot # F-3879) was removed from the-20 ℃ cooler and left at 4 ℃ for 18 hours. The bottle was then removed from the cooler and allowed to warm to room temperature for 60 minutes. 60ml of water at 37 ℃ were added to the bottle and mixed for 15 minutes at 37 ℃. After being in solution, fibrinogen was dialyzed against incomplete fibrinogen buffer (IFB, CFB without HSA and Tween) for 4 hours at room temperature. At the end of four hours, HSA was added to a concentration of 80mg/g total protein, and Tween80 (animal derived) was added to a concentration of 15mg/g total protein. The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5mg/ml using CFB. After preparation, fibrinogen was placed on ice prior to use. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. CTB was used to modulate thrombin concentration in order to deliver the following amounts: 2.5, 0.25, 0.125, 0.083 and 0.0625 units/mg fibrinogen (when mixed) which corresponds to 624, 62.4, 31.2, 20.8 and 15.6 units/ml thrombin prior to mixing. After preparation, thrombin was placed on ice prior to use. The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. A 3ml syringe fitted with an 18 gauge needle was filled with 2ml of fibrinogen and a 1ml second syringe fitted with a22 gauge needle was filled with 0.3ml of thrombin. The contents of both syringes were dispensed simultaneously into each mold. After filling, the mold was placed on top of the liquid nitrogen for 30 seconds, then returned to the-80 ℃ cooler for at least 2 hours before being placed in the freeze-dryer. They were then lyophilized as described below and tested for performance using the EVPA and adhesion tests as described below. These results are shown in fig. 4A and 4B. Example 10

The liner material was placed in a 2.4X2.4cm PVC mold. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. A second sheet of PETG plastic is cut to fit on top of the mold and held in place by clips at each end of the mold, creating a closed mold. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes. Fibrinogen was formulated in CFB (ERL, lot 3060). The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5mg/ml using CFB. After preparation, fibrinogen was placed on ice prior to use. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. CTB was used to modulate thrombin concentration in order to deliver the following amounts: 2.5, 0.25, 0.125, 0.083 and 0.062 units/mg fibrinogen (after mixing) which corresponds to 624, 62.4, 31.2, 20.8 and 15.6 units/ml thrombin prior to mixing. After preparation, thrombin was placed on ice prior to use. The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. A 3ml syringe fitted with an 18 gauge needle was filled with 2ml of fibrinogen and a 1ml second syringe fitted with a22 gauge needle was filled with 0.3ml of thrombin. The contents of both syringes were dispensed simultaneously into each mold. After filling, the mold was placed on top of the liquid nitrogen for 30 seconds, then returned to the-80 ℃ cooler for at least 2 hours before being placed in the freeze-dryer. They were then lyophilized as described below and tested for performance using the EVPA and adhesion tests as described below. These results are shown in fig. 4A and 4B. Example 11

The liner material was placed in a 2.4X2.4cm PVC mold. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. A second piece of PETG plastic was cut to fit on top of the 2.4X2.4 mold and held in place by using clips at each end of the mold, creating a closed mold. The mold was then placed in a-80 ℃ cooler for at least 60 minutes. Vials containing 3 grams of fibrinogen (Sigma Lot # F-3879) were removed from the-20 ℃ cooler and placed at 4 ℃ for 18 hours. The bottle was then removed from the cooler and allowed to warm to room temperature for 60 minutes. 60ml of water at 37 ℃ were added to the bottle and mixed for 15 minutes at 37 ℃. After being in solution, fibrinogen was dialyzed against IFB. At the end of four hours, HSA was added to a concentration of 80mg/g total protein, and Tween80 (animal derived) was added to a concentration of 15mg/g total protein. The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5mg/ml using CFB. After preparation, fibrinogen was placed on ice prior to use. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin concentration was adjusted so as to deliver the following amounts: 2.5, 0.25, 0.125, 0.1 and 0.083 units per mg of fibrinogen (when mixed) which corresponds to 624, 62.4, 31.2, 24.96 and 20.79 units per ml of thrombin prior to mixing. After preparation, thrombin was placed on ice prior to use. The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was then removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. A 3ml syringe fitted with an 18 gauge needle was filled with 2ml of fibrinogen and a 1ml second syringe fitted with a22 gauge needle was filled with 0.3ml of thrombin. The contents of both syringes were dispensed simultaneously into each mold. After filling, the mold was placed on top of the liquid nitrogen for 30 seconds, then returned to the-80 ℃ cooler for at least 2 hours before being placed in the freeze-dryer. They were then lyophilized as described below and tested for performance using the EVPA and adhesion tests as described below. These results are shown in fig. 4A and 4B. Example 12

The liner material was placed in a 2.4X2.4cm PVC mold. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. A second sheet of PETG plastic is cut to fit on top of the mold and held in place by using clips at each end of the mold, creating a closed mold. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes.

Vials containing 3 grams of fibrinogen (Sigma, lot number F-3879) were removed from the-20 ℃ cooler and placed at 4 ℃ for 18 hours. The bottle was then allowed to warm to room temperature for 60 minutes. 60ml of water at 37 ℃ were added to the bottle and mixed for 20 minutes at 37 ℃. After being in solution, fibrinogen was dialyzed against IFB. At the end of four hours, Human Serum Albumin (HSA) was added to a concentration of 80mg/g total protein, and Tween80 (animal derived) was added to a concentration of 15mg/g total protein. The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5mg/ml using CFB. After preparation, fibrinogen was placed on ice prior to use.

Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin concentration was adjusted so as to deliver the following amounts: 2.5, 0.25, 0.125, 0.08 and 0.06 units/mg fibrinogen (after mixing) which corresponds to 624, 62.4, 31.2, 20.8 and 15.6 units/ml thrombin prior to mixing. After preparation, thrombin was placed on ice prior to use. The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. A 3ml syringe fitted with an 18 gauge needle was filled with 2ml of fibrinogen and a 1ml second syringe fitted with a22 gauge needle was filled with 0.3ml of thrombin. The contents of both syringes were dispensed simultaneously into each mold. After filling, the mold was placed on top of the liquid nitrogen for 30 seconds, then returned to the-80 ℃ cooler for at least 2 hours before being placed in the freeze-dryer. They were then lyophilized as described below and tested for performance using the EVPA and adhesion tests as described below. Three-layer (interlayer) dressing

Using the method described in us patent 6,762,336, a trilayer dressing was produced using the same sources of fibrinogen and thrombin as the monolithic dressing described above was produced. Results

The results of the EVPA and adhesion tests are shown in fig. 4A and 4B, respectively. Conclusion (examples 6-12):

dressings produced using 2.5 to 0.025 thrombin units/mg of fibrinogen were effective in both trials, while those with greater or lesser thrombin to fibrinogen ratios were not. Significantly greater activity was observed in the range of 2.5 to 0.05 thrombin units/mg fibrinogen. Greatly improved performance was observed in the range of 0.25 to 0.062 thrombin units/mg fibrinogen, while the best performance was observed in the range of 0.125 to 0.08 thrombin units/mg fibrinogen. This is in contrast to dressings produced using the method described in us 6,762,336 which give full performance at 12.5 thrombin units/mg fibrinogen, produce unacceptable performance when the thrombin concentration is reduced to below 12.5 thrombin units/mg fibrinogen and retain virtually no activity at 1.4 thrombin units/mg fibrinogen. This difference in both the limits and the optimum level of performance is more profound given that the performance of the tri-layer dressing of us patent 6,762,336 decreases with the use of reduced amounts of thrombin and that the dressings described herein exhibit increased activity in this range. Example 13

The liner material was cut and placed in a 2.4X2.4cm per PETG die. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes. Enzyme Research Laboratories (ERL) fibrinogen was formulated in CFB, lot number 3114. The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin was adjusted with CTB to deliver 0.1 units/mg fibrinogen or 25 units/ml thrombin. After preparation, thrombin was placed on ice prior to use. The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate placed on dry ice. The repeat pipettor was filled with fibrinogen and the second repeat pipettor was filled with thrombin. 2ml of fibrinogen and 300 microliters of thrombin were simultaneously dispensed into each mold. After filling the molds with reagents, they were returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. One set of dressings was freeze dried on day 0, while the remaining dressings were stored frozen at-80 ℃. The second set of dressings were freeze-dried on day 7 and the third set of dressings were freeze-dried on the fourteenth day.

After all dressings had been freeze dried, they were tested using the EVPA, adhesion, and weight tests described herein. As a result:

days of freezing before lyophilization	EVPA # PASS/TOTAL	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						0	5/6	3.5	0.5	168.0	63.2
7	6/6	3.8	0.4	164.7	29.4
						14	6/6	3.7	0.5	139.7	39.7

These results are also graphically represented in fig. 5A and 5B. And (4) conclusion:

the well-mixed frozen fibrinogen and thrombin combination remained stable and functional for 7 and 14 days without significant degradation of performance. Longer storage times are expected to produce similar results. Example 14

The liner material was cut and placed in a 2.4X2.4cm per PETG die. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes.

Dressing set 1 (albumin free, Tween80 free): enzyme Research Laboratories (ERL) fibrinogen was formulated in 100mM sodium chloride, 1.1mM calcium chloride, 10mM Tris, 10mM sodium citrate, and 1.5% sucrose, lot 3130. The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml.

Dressing set 2 (albumin-free, Tween 80-containing): ERL fibrinogen was formulated in 100mM sodium chloride, 1.1mM calcium chloride, 10mM Tris, 10mM sodium citrate, and 1.5% sucrose. Tween80 (of animal origin) was added to 15mg/g total protein. The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml.

Dressing set 3 (albumin-containing, Tween 80-free): ERL fibrinogen was formulated in 100mM sodium chloride, 1.1mM calcium chloride, 10mM Tris, 10mM sodium citrate, and 1.5% sucrose. HSA was added to 80mg/g total protein. The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml.

Dressing set 4 (albumin-containing, Tween 80-containing): ERL fibrinogen was formulated in 100mM sodium chloride, 1.1mM calcium chloride, 10mM Tris, 10mM sodium citrate, and 1.5% sucrose (fibrinogen complete buffer). In addition, HSA was added to 80mg/g total protein and Tween80 (animal derived) to 15mg/g total protein. The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, the fibrinogen solution was placed on ice prior to use.

Thrombin was prepared in 150mM sodium chloride, 40mM calcium chloride, 10mM Tris and 100mM L-lysine, plus 100. mu.g/ml HSA. The final pH of thrombin was 7.4. + -. 0.1. Thrombin was adjusted to deliver 0.1 units/mg fibrinogen or 25 units/ml thrombin. After preparation, the thrombin solution was placed on ice prior to use.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate placed on dry ice. The repeat pipettor was filled with fibrinogen solution and the second repeat pipettor was filled with thrombin solution. 2ml of fibrinogen solution and 300 microliters of thrombin solution were simultaneously dispensed into each mold. After filling the molds with reagents, they were returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. KnotAnd (4) fruit:

preparation	EVPA # PASS/TOTAL	Adhesion of	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						-Alb-Tween	0/6	0.8	1.0	24.0	26.3
-Alb+Tween	3/6	3.3	0.8	114.7	40.8
						+Alb-Tween	V6	1.7	1.0	45.0	39.9
+Alb+Tween	5/6	3.5	0.5	131.3	32.0

And (4) conclusion:

the results show that the addition of albumin improves dressing performance. The addition of Tween even further improved the performance. The combination of the two yields the best performance. Example 15

The die consisted of a pair of aluminum plates separated by a plastic spacer of 3/16 "Plexiglas having 1" x1 cut therein"of a square cut. In use, the open side of the cut is directed to the top of a vertically mounted plate-spacer-plate "sandwich" which together form a mould for the dressing. The mold was pre-cooled by dipping it in dry ice pellets, with the top slightly above the dry ice. The gasket material is then cut and placed in each mold. ERL fibrinogen was formulated in CFB. The final pH of fibrinogen was 7.4. + -. 0.1. Adjusting fibrinogen concentration to produce a final product having 13mg/cm²A dressing of fibrinogen. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin was adjusted to deliver 0.1 units/mg fibrinogen in the final dressing.

The fibrinogen and thrombin were cooled to the desired temperature (2, 4,6 and 8 ℃) and mixed in a pre-cooled 15mL conical tube and mixed using a high speed vortex for 5 seconds before dispensing into the mold. The fibrinogen-thrombin mixture was then pipetted into the mold using a serological pipette. As a result:and (4) conclusion:

functional dressings were made that included 38-57% free alpha chain and no gamma-gamma dimer. Example 16

The mold consists of a pair of aluminum or steel plates separated by an 3/16 "Plexiglas plastic spacer having a 1" x1 "square cut therein. In use, the open side of the cut is directed to the top of a vertically mounted plate-spacer-plate "sandwich" which together form a mould for the dressing. In some groups, a thin Plastic (PVC) liner is used to provide the product contact surface for the mold cooling plates. The molds are pre-cooled to the desired temperature, and the gasket material is cut and placed in each mold.

ERL fibrinogen was formulated in CFB. The final pH of fibrinogen was 7.4. + -. 0.1. Modulating fibrinogen concentration for productionHas 13mg/cm in the final product²A dressing of fibrinogen. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin was adjusted to deliver 0.1 units/mg fibrinogen in the final dressing.

Fibrinogen and thrombin were dispensed into the mold. After filling the molds with reagents, they were frozen, then held at-80 ℃ and then placed in a freeze-dryer. The dressing was then lyophilized as described above. After completion, the dressing was stored in a low moisture transfer foil pouch containing 5 grams of desiccant.

The dressing was then evaluated for performance in terms of EVPA, adhesion test, as previously described. The produced dressing was biochemically characterized by gel electrophoresis testing as described above. As a result:and (4) conclusion:

the produced dressing passed all performance tests. The percentage of free alpha chains was 46-56% while there were no detectable gamma-gamma dimers in any dressing. Example 17

The die consisted of a pair of aluminum plates separated by an 3/16 "Plexiglas plastic spacer having a 1" x1 "square cut therein. In use, the open side of the cut is directed to the top of a vertically mounted plate-spacer-plate "sandwich" which together form a mould for the dressing. The mold was pre-cooled by dipping it in dry ice pellets, with the top slightly above the dry ice. The gasket material is then cut and placed in each mold. ERL fibrinogen was formulated in CFB. The final pH of fibrinogen was 7.4. + -. 0.1. Adjusting fibrinogen concentration to produce a final product having 13mg/cm²A dressing of fibrinogen. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin was adjusted to deliver 0.1 units/mg fibrinogen in the final dressing.

Fibrinogen and thrombin were kept on ice and the following operations were performed: add to a pre-cooled 15mL conical tube and mix for 5 seconds using a high speed vortex and then pipette into a mold using a serological pipettor; or in a 60mL syringe for dispensing fibrinogen and a 3mL syringe for dispensing thrombin, the syringes were placed in a linear syringe pump, and each syringe was connected to the opposite end of the T-connector using tubing. The pump is then activated and the fibrinogen-thrombin mixture is dispensed into the mold using the bottom of the connector.

After filling the molds with reagents, they were frozen, then held at-80 ℃ and then placed in a freeze-dryer. The dressing was then lyophilized as described above. After completion, the dressing was stored in a low moisture transfer foil pouch containing 5 grams of desiccant.

the produced dressing failed all performance tests. The free alpha level in these dressings was 100%, indicating complete conversion of the original fibrinogen to fibrin Ia. The gamma-gamma dimer level was 55-58%. There was no difference between the different methods used to mix fibrinogen and thrombin. Example 18

The liner material was cut and placed in each 1.5X1.5cm PVC mold. 15 microliters of 2% sucrose was pipetted onto the top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes. Fibrinogen was formulated in CFB (ERL, lot 3112). The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin was adjusted to deliver the following amounts: 0.1 units/mg fibrinogen or 25.0 units/ml thrombin. After preparation, thrombin was placed on ice prior to use. The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃.

The mold was removed from the-80 ℃ cooler and placed on an aluminum plate placed on dry ice. 2.4ml of fibrinogen was pipetted into a 12X75mm, pre-cooled tube, followed by 360. mu.l of thrombin addition. The tube was vortexed for 3 seconds, and an 897 μ Ι aliquot was removed and pipetted into the mold. Six time points (25 seconds, 1, 2,3, 5, and 8 minutes) and one control dressing were produced. The control dressing was not pre-mixed and 780 μ l fibrinogen and 117 μ l thrombin were pipetted simultaneously into the molds on dry ice. After filling each mold with reagents, they were returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. As a result:nt: the conclusion of the test is not carried out:

fully functional dressing example 19 with free alpha levels of 0-42% was produced

Enzyme Research Laboratories (ERL) fibrinogen was formulated in CFB, lot number 3112. The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use.

Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin was adjusted to deliver 0.1 units/mg fibrinogen or 25 units/ml thrombin. After preparation, thrombin was placed on ice prior to use.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The molds were removed from the 80 ℃ cooler and several groups were placed on steel freeze dryer shelves. Shelf temperatures were as follows: -10 ℃, -20 ℃, -30 ℃ and-40 ℃ and the mould was placed on a stand for 30 minutes for equilibration. A 3m l syringe fitted with an 18 gauge needle was filled with 2ml of fibrinogen and a 1ml second syringe fitted with a22 gauge needle was filled with 0.3ml of thrombin. The contents of both syringes were dispensed simultaneously into each mold. After filling the molds with the reagents, they were kept inside the freeze-dryer at the set temperature for 30 minutes. They were then returned to the-80 ℃ cooler for at least 2 hours before freeze-drying as described previously.

In addition, 2 sets of dressings were produced by placing on an aluminum plate placed on top of dry ice. A 3ml syringe fitted with an 18 gauge needle was filled with 2ml of fibrinogen and a 1ml second syringe fitted with a22 gauge needle was filled with 0.3ml of thrombin. The contents of both syringes were dispensed simultaneously into each mold. After filling the molds with the reagents, they were kept on dry ice for 5 minutes or placed on liquid nitrogen for 30 seconds. They were then returned to the-80 ℃ cooler for at least 2 hours before freeze-drying as described previously.

The lyophilized dressing was tested for performance in terms of EVPA, adhesion and weight tests. In addition, they were analyzed using the gel electrophoresis test described. As a result:and (4) conclusion:

the dressings produced had a free alpha chain level of 10% to 33% and no detectable gamma-gamma dimer. The performance is inversely related to the level of free alpha chain and to the freezing temperature. Example 20

Human fibrinogen (Sigma, st.louis) was formulated in CFB at a concentration of 35mg fibrinogen/ml and bovine thrombin (Sigma, st.louis) was formulated in CTB at 87.5U/ml. The pH of the buffer was adjusted to fit the target pH of each well.

Both fibrinogen and thrombin had a pH range of 5.5-8.5 in 0.5 increments. Two test temperatures, 4 ℃ and 24 ℃, were used. The experiment was performed in flat-bottom 96-well ELISA plates (Nalgene, VWR).

Mu.l of fibrinogen was pipetted into each well of a 96-well plate (3.5 mg/well) followed by 100. mu.l of thrombin (8.75U/well). See the plate arrangement below. The plates were incubated for 10 minutes before evaluation. Clot formation and structure were evaluated by inversion to detect clot formation and adhesion, and opacity. The arrangement of the plate:as a result: room temperature panel:

clots formed in all wells. Fibrinogen at pH5.5 and 6.0 had very opaque clots at all pH ranges of thrombin. Fibrinogen of pH6.5 has an opaque clot when thrombin of pH 5.5-7.0 is used. At thrombin above pH 7.5, the clot is clear. Fibrinogen with pH levels of 7.0 and higher forms a clear clot at all pH levels of thrombin. The results are shown in FIG. 6A. 4 ℃ plate:

clots formed in all wells. Fibrinogen at pH5.5 and 6.0 had very opaque clots at all pH ranges of thrombin. Fibrinogen of pH6.5 has an opaque clot when thrombin of pH 5.5-7.0 is used. At thrombin above pH 7.5, the clot is clear. Fibrinogen with pH levels of 7.0 and higher forms a clear clot at all pH levels of thrombin. The results are shown in FIG. 6B. Adhesion:

clot adhesion was determined by inverting the plate over a paper towel and tapping the back of the plate gently. After 30 seconds the plate was removed and the clot retained in the plate was considered to be sticky, while the clot dropped on the paper towel was considered to be non-sticky.

Although clots differ in their opacity, clots formed using fibrinogen at pH5.5 and 6.0 lack adhesiveness. This is true for fibrinogen with pH6.5 at thrombin pH 5.5-7.0. Once the fibrinogen pH was 7.0 or greater, a viscous clot formed over all of the thrombin pH ranges tested. This is the case for both room temperature and 4 ℃ plates. And (4) conclusion:

from these results it was determined that the dressing preferably has fibrinogen with a pH of 7.0 or greater, but that the pH of thrombin can vary from 5.5 to 8.5. Example 21

Human fibrinogen (Sigma, st.louis) was formulated in modified CFB (without calcium chloride) at a concentration of 35mg fibrinogen/ml and bovine thrombin (Sigma, st.louis) was formulated in modified CTB (without calcium chloride) at a concentration of 87.5U/ml. The pH of the buffer was adjusted to fit the target pH of each well.

clots formed in all wells. Fibrinogen at pH5.5 and 6.0 had very opaque clots at all pH ranges of thrombin. The fibrinogen of pH6.5 did not opacify the clot when thrombin of pH 5.5-8.5 was used, unlike previous results. The clot is clear with thrombin at a pH above 7.5. Fibrinogen with pH levels of 6.5 and higher forms clots at all pH levels of thrombin. The results are shown in FIG. 6C. Plate at 4 ℃:

clots formed in all wells. Fibrinogen at pH5.5 and 6.0 had very opaque clots at all pH ranges of thrombin. Fibrinogen of pH6.5 has an opaque clot when thrombin of pH 5.5-7.0 is used. At thrombin above pH 7.5, the clot is clear. Fibrinogen with pH levels of 7.0 and higher forms clots at all pH levels of thrombin. The results are shown in fig. 6D. Adhesion:

clot adhesion was determined by inverting the plate over a paper towel and tapping the back of the plate gently. After 30 seconds the plate was removed and the clot retained in the plate as being coherent, while the clot that fell on the paper towel was considered non-coherent. And (4) observing results:

although clots differ in their opacity, clots formed using fibrinogen of ph5.5 and 6.0 lack adhesiveness. Once the pH of fibrinogen is 6.5 or greater, a viscous clot forms at all thrombin pH ranges tested. This is the case for both room temperature and 4 ℃ plates. These results are comparable to the results obtained with the buffer without CaC₁₂The previous results of the formation of a viscous clot with fibrinogen at pH6.5 differed. And (4) conclusion:

from these results, it was confirmed that there is no CaC₁₂The fibrinogen of the dressing should have a pH of 6.5 or greater in the presence of (a), but the pH of thrombin may vary from at least 5.5 to 8.5. Example 22

Human fibrinogen (Sigma, st. louis) was formulated in CFB at a concentration of 35mg fibrinogen/ml. Bovine thrombin (Sigma, St. Louis) was prepared at 87.5U/ml in CTB. The pH of the buffer was adjusted to fit the target pH of each well. The dressings were produced in a disposable 2.4X2.4cm histological mould. An absorbable padding material. Syringe (2.0 ml). Freezing vertically and bidirectionally on dry ice.

The liner material was placed in each 2.4X2.4cm PVC mold. 15 microliters of 2% sucrose was pipetted onto the top of each of the four corners of the liner material. A second sheet of PETG plastic was fitted on top of the 1.5X1.5 mold and fixed in place. This forms a closed mold. The mold was then placed in a-80 ℃ cooler for at least 60 minutes.

Fibrinogen (Sigma) was formulated in CFB. The fibrinogen concentration was adjusted to 37.5mg/ml using CFB. The final pH of fibrinogen was adjusted as shown below. After preparation, fibrinogen was placed on ice prior to use.

Thrombin was formulated in CTB. The final pH of thrombin was adjusted as shown below. The thrombin concentration was adjusted using CTB so as to deliver 0.1 units/mg fibrinogen (when mixed), which corresponds to 25 units/ml thrombin prior to mixing. After preparation, thrombin was placed on ice prior to use.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was then removed from the-80 ℃ cooler and placed between pre-cooled aluminum plates on dry ice. A 18 gauge needle was used to punch 3 holes in the top of the mold. One well was used for injection of fibrinogen and the second for injection of thrombin. These holes are located at opposite ends of the mold, while the third hole is located in the center of the top of the mold and serves as a vent to release air displaced from the interior of the mold. Two 3ml syringes were then filled with 2.0ml of fibrinogen and thrombin, respectively. They are then injected simultaneously into the mold through two holes in the ends of the mold. After filling the molds with the reagents, the molds were covered with dry ice pellets and allowed to freeze for 2 minutes before returning them to the-80 ℃ cooler for at least two hours before being placed in the freeze-dryer. They were then lyophilized as described below. Production of a combination of dressings:

dressing number	Fibrinogen pH	Thrombin pH
			1	5.5	6.0
2	6.0	6.5
			3	6.5	8.0
4	7.0	7.0
			5	7.5	7.0
6	8.0	7.5
			7	8.5	7.5

Evaluation criteria

The dressing was evaluated for appearance. It has been determined through extensive experimentation to produce dressings based on fibrin sealant that it appears that dressings made from bulk powdered materials perform poorly. Similarly, visual inspection of preformed fibrin in the dressing correlates inversely with performance, as this indicates that the dressing has poor adherence. The ease and rate of hydration were also easily evaluated, predicting dressing performance that hydrates easily and quickly in a uniform manner. Finally, the ability to form a clot consisting of dense and uniform fibrin is also desirable for good performance and can be evaluated visually.

All dressings were evaluated visually. In addition, dressings 1-4 and 6 were hydrated with 2ml of water at 37 ℃ and the rate of hydration, the ability to form clots, and their subsequent adhesiveness were evaluated visually. As a result: appearance after freeze-drying:

all dressings were easily evaluated by visual criteria (see fig. 7A and 7B). Dressings 1 and 2 were very powdered relative to the other dressings. The relative proportion of dressing of the powder decreases with increasing pH of the fibrinogen.

Dressings 1 and 2 have some preformed fibrin, which can be seen in the following figures, as dense white mottled areas, while dressings 3, 4 and 6 have a large amount of preformed fibrin and dissolve slowly. In contrast, dressing No. 5 had high integrity and no visually detectable pre-formed fibrin. These results are summarized in the following table.

Dressing number	Fibrinogen pH	Thrombin pH	Integrity of	Preformed fibrin	Rate of hydration
						1	5.5	6.0	Is very powdered	Is that	Medium and high grade
2	6.0	6.5	Powdering into powder	Same as above	Same as above
						3	6.5	8.0	Good taste	Large scale of	Difficulty and slowness
4	7.0	7.0	Is excellent in	Is that	Same as above
						5	7.5	7.0	Same as above	Whether or not	Not tested
6	8.0	7.5	Same as above	Large scale of	Difficulty and slowness
						7	8.5	7.5	Same as above	Is that	Not tested

Conclusion

The dressing characteristics are given in the table above. All dressings formed clots in all parts of the pH range. Those with fibrinogen pH of 5.5-6.0 and thrombin of 6.0-6.5 have minimal integrity prior to hydration. They also form small amounts of fibrin prior to hydration. Those with fibrinogen pH of 6.5-7.0 and thrombin pH of 8.0-7.0 have greater integrity, but more fibrin before hydration forms. Dressings made with 8.0-8.5 fibrinogen and 7.5 thrombin had good integrity, but showed significant fibrin formation prior to hydration and were difficult to hydrate. In contrast, at a pH of 7.0 for both fibrinogen and thrombin, the resulting dressing has excellent integrity and a smaller amount of pre-formed fibrin. The use of a combination of fibrinogen at pH 7.5 and thrombin at pH7.0 resulted in an optimal dressing. Example 23

The gasket material (DEXON) was cut and placed in each 1.5X1.5cm PVC mold. 15 microliters of 2% sucrose was pipetted onto the top of each of the four corners of the liner material. A second piece of PETG plastic was cut to fit on top of the 1.5X1.5 mold and held in place by the addition of clips located at each end of the mold. This closes the mold. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes.

Fibrinogen was formulated in CFB (batch 3100). The final pH of fibrinogen was 7.4. + -. 0.1. Fibrinogen concentration was adjusted to 37.5mg/ml with CFB. After preparation, fibrinogen was placed on ice prior to use.

Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin was adjusted with CTB to deliver 0.1 units/mg fibrinogen or 25 units/ml thrombin. After preparation, thrombin was placed on ice prior to use.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed between two aluminum plates in an upright (vertical) position and then placed on dry ice. A 18 gauge needle was used to punch 3 holes in the top of the mold. One well serves as an inlet for fibrinogen, a second well serves as an inlet for thrombin, and a third well serves to release any air in the mold. One pipette was filled with fibrinogen and the second pipette was filled with thrombin. 0.78ml of fibrinogen and 0.17ml of thrombin were dispensed simultaneously into each mould. After filling each mold with reagents, the molds were placed on top of liquid nitrogen for 30 seconds, then returned to a-80 ℃ cooler for at least 2 hours before being placed in a freeze-dryer and lyophilized as described previously. As a result:

group of	EVPA # PASS/COUNT	Adhesion test score (mean. + -. standard deviation)
			Closed/upright	5/5	3.4+0.5

And (4) conclusion:

from these results it was determined that dressings produced using an overall pH of 7.3-7.5 yielded excellent performance. Example 24

For dressings using a liner, the liner material was cut and placed in 2.4X2.4cm moulds per PETG. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes.

Fibrinogen was formulated in CFB (lot 3114). The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on a copper plate placed on dry ice. The repeat pipettor was filled with fibrinogen and the second repeat pipettor was filled with thrombin. 2ml of fibrinogen and 300 microliters of thrombin were simultaneously dispensed into each mold. After filling the molds with reagents, they were returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. The dressing was then lyophilized as described. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						Open/horizontal	6/6	3.7	0.5	153	37.3

Example 25

The gasket material was placed in each 1.5X1.5cm PVC mold. 15 microliters of 2% sucrose was pipetted onto the top of each of the four corners of the liner material. A second sheet of PETG plastic was fitted on top of the 1.5X1.5 mold and fixed in place. This forms a closed mold. The mold was then placed in a-80 ℃ cooler for at least 60 minutes.

Fibrinogen was formulated in CFB (ERL, batch 3100). The fibrinogen concentration was adjusted to 37.5mg/ml using CFB. The final pH of fibrinogen was 7.4. + -. 0.1. After preparation, fibrinogen was placed on ice prior to use.

Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. The thrombin concentration was adjusted with CTB so as to deliver 0.1 units/mg fibrinogen (when mixed), which corresponds to 25 units/ml thrombin prior to mixing. After preparation, thrombin was placed on ice prior to use.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. A 18 gauge needle was used to punch 3 holes in the top of the mold. One hole was used for injection of fibrinogen, a second for injection of thrombin and a third hole was used as a vent to release air displaced from the interior of the mold. The pipettor was then filled with fibrinogen and the second pipettor was filled with thrombin. 0.78ml of fibrinogen and 0.17ml of thrombin were simultaneously injected into each mold by these pipettors. After filling, the mold was placed on top of the liquid nitrogen bathLeft for 30 seconds and then returned to the-80 ℃ cooler for at least 2 hours before being placed in the freeze-dryer. They were then lyophilized as described. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion
				Closed/upright	5/5	3.4	0.5

Example 26

For all dressings, ERL fibrinogen, lot 3130 was formulated in CFB. The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use.

Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin was adjusted to deliver 0.1 units/mg fibrinogen or 25 units/ml thrombin. For the group with the shredded Vicryl mesh dispersed inside, this support material was cut into pieces of about 1mmx1mm and dispersed in a thrombin solution before filling the mold. After preparation, thrombin was placed on ice prior to use.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. A cylindrical mold made of a 10mL or 3mL polypropylene syringe (Becton Dickinson) with the Luer lock cuff terminating end removed was used. The piston was withdrawn to 6mL and 2mL marks, respectively. For dressings utilizing pads, the support material is cut and placed in each mold and pressed downward until it abuts the piston. After preparation, the mold was placed upright and surrounded with dry ice, leaving a riot on the topAn exposed opening. 1mL of fibrinogen and 0.15mL of thrombin (with or without liner material dispersed inside) were dispensed into a 10mL mold, and 1mL of fibrinogen and 0.15mL of thrombin (with or without liner material dispersed inside) were dispensed into a 3mL mold and allowed to freeze for 5 minutes. The molds were then placed in a-80 ℃ cooler for at least 2 hours before being placed in a freeze-dryer and lyophilized as described. As a result:

group of	EVPA # PASS/COUNT
		Tubular (Syringe)	3/3

Small knot

Group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						Open/horizontal	6/6	3.7	0.5	153	37.3
Closed/upright	5/5	3.4	0.5
						Tubular (Syringe)	3/3

And (4) conclusion:

the produced dressing passed the EVPA used for the production conditions. Example 27

The gasket material was placed in each 1.5X1.5cm PVC mold. 15 microliters of 2% sucrose was pipetted onto the top of each of the four corners of the liner material. A second sheet of PETG plastic was fitted on top of the 1.5X1.5 mold and fixed in place. This forms a closed mould. The mold was then placed in a-80 ℃ cooler for at least 60 minutes.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. A 18 gauge needle was used to punch 3 holes in the top of the mold. One hole was used for injection of fibrinogen, a second for injection of thrombin and a third hole was used as a vent to release air displaced from the interior of the mold. The pipettor was then filled with fibrinogen and the second pipettor was filled with thrombin. 0.78ml of fibrinogen and 0.17ml of thrombin were simultaneously injected into each mold by these pipettors. After filling, the mold was placed on top of a liquid nitrogen bath for 30 seconds, then returned to a-80 ℃ cooler for at least 2 hours before being placed in a freeze-dryer. They were then lyophilized as described. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion
				Liquid transfer device	5/5	3.4	0.5

Example 28

For dressings using a liner, the liner material was cut and placed in each petg2.4x2.4cm mold. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes.

ERL fibrinogen was formulated in CFB (lot 3114). The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on a copper plate, which was placed on top of dry ice. The repeat pipettor was filled with fibrinogen and the second repeat pipettor was filled with thrombin. 2ml of fibrinogen and 300 microliters of thrombin were simultaneously dispensed into each mold. After filling the molds with reagents, they were returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. The dressing was then lyophilized as described.As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						Repeat pipettor	6/6	3.7	0.5	153	37.3

Example 29

The liner material was placed in a 2.4X2.4cm PVC mold. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. A 3ml syringe fitted with an 18 gauge needle was filled with 2ml of fibrinogen,and a 1ml second syringe fitted with a22 gauge needle was filled with 0.3ml thrombin. The contents of both syringes were dispensed simultaneously into each mold. After filling, the mold was placed on top of the liquid nitrogen for 30 seconds, then returned to the-80 ℃ cooler for at least 2 hours before being placed in the freeze-dryer. They were then lyophilized as described. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion
				Syringe with a needle	4/4	3.8	0.5

Example 30

The liner material was cut and placed in each PETG 10X10cm mold. 50 microliters of 2% sucrose was pipetted onto the top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes.

Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin was adjusted to deliver 0.1 units/mg fibrinogen or 25 units/ml thrombin. After preparation, thrombin was placed on ice prior to use. Thrombin was adjusted to deliver 0.1 units/mg fibrinogen or 25 units/ml thrombin. After preparation, thrombin was placed on ice prior to use.

Fibrinogen and clotting prior to dispensingThe temperature of the enzyme was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate, which was placed on top of the dry ice. The aluminum plate had a 0.25 inch hole drilled in the middle and a fitting was attached so that a length of tubing could be connected to a vacuum source. The mold was centered over the hole in the aluminum plate and the vacuum was turned on. Vacuum has two functions: preventing the mold from moving and keeping it flat with respect to the aluminum plate. 35 ml of fibrinogen and 5.25 ml of thrombin were placed in a 50ml tube, inverted three times and poured into a mold. After filling the molds and applying the support material as described above, they were returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. The dressing was then lyophilized as described. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						Mixing and pouring	6/6	3.8	0.4	163	31.5

Example 31

The liner material was cut and placed in each 1.5X1.5cm PVC mold. 15 microliters of 2% sucrose was pipetted onto the top of each of the four corners of the liner material. The mold was placed in a-80 ℃ cooler for at least 60 minutes.

ERL fibrinogen was formulated in CFB (batch No. 2890). The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use.

Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. Thrombin was adjusted to deliver 0.1 units/mg fibrinogen or 25 units/ml thrombin. After preparation, thrombin was placed on ice prior to use. Thrombin was adjusted to deliver 0.1 units/mg fibrinogen or 25 units/ml thrombin.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate placed on dry ice. The two automatic dispensing systems from IJ fisher were combined according to the manufacturer's instructions. One syringe was filled with fibrinogen and the second syringe was filled with thrombin. Simultaneously, 0.78ml of fibrinogen and 0.17ml of thrombin were dispensed into each mold. After filling, the mold was placed on top of the liquid nitrogen for 30 seconds, then returned to the-80 ℃ cooler for at least 2 hours before being placed in the freeze-dryer. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion
				Dispenser	5/5	4.0	0.0

And (3) knotting:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						Liquid transfer device	5/5	3.4	0.5
Repeat pipettor	6/6	3.7	0.5	153	37.3
						Syringe with a needle	4/4	3.8	0.5
Mixing and pouring	6/6	3.8	0.4	163	31.5
						Dispenser	5/5	4.0	0.0

And (4) conclusion:

dressings produced using a variety of filling techniques passed the tests performed on them. This indicates that the thrombin and fibrinogen components produce an acceptable mix in the case of the various filling methods. Example 32

Fibrinogen ERL was formulated in CFB (lot 3114). The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on a copper plate, which was placed on top of dry ice. The repeat pipettor was filled with fibrinogen and the second repeat pipettor was filled with thrombin. 2ml of fibrinogen and 300 microliters of thrombin were simultaneously dispensed into each mold. After filling the molds with reagents, they were returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. The dressing was then lyophilized as described. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						Unidirectional	6/6	3.7	0.5	153	37.3

Example 33

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. A 18 gauge needle was used to punch 3 holes in the top of the mold. One hole was used for injection of fibrinogen, a second for injection of thrombin and a third hole was used as a vent to release air displaced from the interior of the mold. The pipettor was then filled with fibrinogen and the second pipettor was filled with thrombin. 0.78ml of fibrinogen and 0.17ml of thrombin were simultaneously injected into each mold by these pipettors. After filling, the mold was placed on top of a liquid nitrogen bath for 30 seconds and then returned to a-80 ℃ cooler for at least 2 hours before being placed in a freeze dryer. They were then lyophilized as described. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion
				Bidirectional	5/5	3.4	0.5

Example 34

ERL fibrinogen was formulated in CFB (run 3130). The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. A cylindrical mold made of a 10mL or 3mL polypropylene syringe (Becton Dickinson) with the Luer lock cuff terminating end removed was used. The piston was withdrawn to 6mL and 2mL marks, respectively. For dressings utilizing pads, the support material is cut and placed in each mold and pressed downward until it abuts the piston. After preparation, the mold was placed upright and surrounded with dry ice, leaving an exposed opening at the top. 1mL of fibrinogen and 0.15mL of thrombin (with or without liner material dispersed inside) were dispensed into a 10mL mold, and 1mL of fibrinogen and 0.15mL of thrombin (with or without liner material dispersed inside) were dispensed into a 3mL mold and allowed to freeze for 5 minutes. The molds were then placed in a-80 ℃ cooler for at least 2 hours before being placed in a freeze-dryer and lyophilized as described. As a result:

group of	EVPA # PASS/COUNT
		Around the surface of the steel pipe	3/3

And (4) conclusion:

dressings produced and frozen under various conditions passed the tests performed for each example. Fibrinogen and thrombin can be combined into a liquid and frozen under various conditions. Example 35

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. A 18 gauge needle was used to punch 3 holes in the top of the mold. One hole was used for injection of fibrinogen, a second for injection of thrombin and a third hole was used as a vent to release air displaced from the interior of the mold. The pipettor was then filled with fibrinogen and the second pipettor was filled with thrombin.0.78ml of fibrinogen and 0.17ml of thrombin were simultaneously injected into each mold by these pipettors. After filling, the mold was placed on top of a liquid nitrogen bath for 30 seconds, then returned to a-80 ℃ cooler for at least 2 hours before being placed in a freeze-dryer. They were then lyophilized as described. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Appearance of the product
					1.5X1.5cm	5/5	3.4	0.5	Smooth, acceptable

Example 36

The liner material was cut and placed in a 2.4X2.4cm per PETG die. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes. ERL fibrinogen was formulated in CFB (lot 3114). The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on a copper plateThe copper plate is located above the dry ice. The repeat pipettor was filled with fibrinogen and the second repeat pipettor was filled with thrombin. 2ml of fibrinogen and 300 microliters of thrombin were simultaneously dispensed into each mold. After filling the molds with reagents, they were returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. The dressing was then lyophilized as described. As a result:

group of

EVPA # PASS/COUNT

Peel test adhesion

Standard deviation of adhesion

Weight maintenance (average) (g)

Standard deviation of weight retention

Appearance of the product

2.4X2.4cm

6/6

3.7

0.5

153

37.3

Smooth, acceptable

Example 37

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate placed on dry ice. The aluminum plate had a 0.25 inch hole drilled in the middle and a fitting was attached so that a length of tubing could be connected to a vacuum source. The mold was centered over the hole in the aluminum plate and the vacuum was turned on. Vacuum has two functions: preventing the mold from moving and keeping it flat with respect to the aluminum plate. 35 ml of fibrinogen and 5.25 ml of thrombin were placed in a 50ml tube, inverted three times and poured into a mold. After filling the molds and applying the support material as described above, they were returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. The dressing was then lyophilized as described. As a result:

group of

EVPA # PASS/COUNT

Peel test adhesion

Standard deviation of adhesion

Weight maintenance (average) (g)

Standard deviation of weight retention

Appearance of the product

10X10cm

6/6

3.8

0.4

163

31.5

Smooth, acceptable

Example 38

The liner material was cut and placed in a 3.7X2.4cm per PETG die. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes.

Fibrinogen was formulated in CFB (ERL, batch 3100). The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate placed on dry ice. One pipette was filled with fibrinogen and the second pipette was filled with thrombin. 3.1ml of fibrinogen and 0.465ml of thrombin were dispensed simultaneously into each mould. After each mold was filled, the molds were placed on top of the liquid nitrogen for 30 seconds, then returned to a-80 ℃ cooler for at least 2 hours before being placed in a freeze dryer. The dressing was then lyophilized as described. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Appearance of the product
					3.7X2.4cm	5/5	3.4	0.5	Smooth, acceptable

Example 39

The liner material was cut and placed in a circular 63.6cm frame²In a mold. 50 microliters of 2% sucrose was pipetted onto the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate placed on dry ice. 22 ml of fibrinogen and 3.3 ml of thrombin were placed in a 50ml tube, inverted three times and poured into a mold. After filling the mold, the mold was placed on top of the liquid nitrogen for 30 seconds, then returned to the-80 ℃ cooler for at least 2 hours before being placed in the freeze-dryer. The dressing was then lyophilized as described. As a result:

group of	Appearance of the product
		63.6cm²	Smooth, acceptable

Example 40

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate placed on dry ice. For a 5.2X5.6cm mold, 10.1 ml of fibrinogen and 1.5 ml of thrombin were placed in a 50ml tube, inverted three times and poured into the mold. A13.0 X8.0cm mold received 36ml of fibrinogen and 5.4ml of thrombin. A7.0 X3.0cm mold received 7.3ml of fibrinogen and 1.1ml of thrombin. After filling the molds with reagents, they were returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. The dressing was then lyophilized as described. As a result:

group of

EVPA # PASS/COUNT

Peel test adhesion

Standard deviation of adhesion

Weight maintenance(average) (g)

Standard deviation of weight retention

Appearance of the product

5.2X5.6cm

Smooth, acceptable

13.0X 8.0cm

8/8

3.3

1.0

99

43

Smooth, acceptable

7.0X3.0cm

3/3

4

0

121

55

Smooth, acceptable

EXAMPLE 41

ERL fibrinogen was formulated in CFB (lot 3112). The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml. After preparation, fibrinogen was placed on ice prior to use.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on dry iceOn an aluminum plate. For a 5.5X6.0cm mold, 11.5 ml of fibrinogen and 1.73 ml of thrombin were placed in a 50ml tube, inverted three times and poured into the mold. A10.0 X8.0cm mold received 27.7ml of fibrinogen and 4.16ml of thrombin. A12.0 X8.0cm mold received 37.4ml of fibrinogen and 5.62ml of thrombin. After each mold was filled with the reagent, it was returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. As a result:

group of	Appearance of the product
		5.5X6.0cm	Smooth, acceptable
10.0X8.0cm	Smooth, acceptable
		12.0X9.0cm	Smooth, acceptable

And (4) conclusion:

the dressing can be produced from small (2.25 cm)²) To large (108 cm)²) Of the different sizes of the components. All dressings produced passed their respective test criteria. The dressing can be produced in any reasonable, useful and acceptable size, provided that the time and temperature of mixing and freezing of the agents is controlled. Example 42

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion
				PVC	5/5	3.4	0.5

Example 43

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						PETG	6/6	3.7	0.5	153	37.3

Example 44

The liner material was cut and placed in each 2.4X2.4cm stainless steel mold. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. The pipettor was then filled with fibrinogen and the second pipettor was filled with thrombin. 2.0ml of fibrinogen and 0.3ml of thrombin were simultaneously injected into each mold by these pipettors. After filling the molds with reagents, they were returned to a-80 ℃ cooler for at least 2 hours and then placed in a freeze-dryer. The dressing was then lyophilized as described. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion
				Stainless steel	3/3	4.0	0.0

Example 45

group of	EVPA # PASS/COUNT
		Polypropylene	3/3

And (3) knotting:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						PVC	5/5	3.4	0.5
PETG	6/6	3.7	0.5	153	37.3
						Stainless steel	3/3	4.0	0.0
Polypropylene	3/3

And (4) conclusion:

various plastics or metals may be used as mould supports to produce the dressing. All of these dressings passed their test standards. Example 46

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						Perforated aluminium	6/6	3.8	0.4	163	31.5

Example 47

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. A 18 gauge needle was used to punch 3 holes in the top of the mold. One hole was used for injection of fibrinogen, a second for injection of thrombin and a third hole was used as a vent to release air displaced from the interior of the mold. The pipettor was then filled with fibrinogen and the second pipettor was filled with thrombin. 0.78ml of fibrinogen and 0.17ml of thrombin were simultaneously injected into each mold by these pipettors. After filling, the mold was placed in liquid nitrogenThe top of the cell was allowed to sit for 30 seconds and then returned to the-80 ℃ cooler for at least 2 hours before being placed in the freeze-dryer. They were then lyophilized as described. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion
				Aluminium	5/5	3.4	0.5

Example 48

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on a copper plate, which was placed on top of dry ice. The repeat pipettor was filled with fibrinogen and the second repeat pipettor was filled with thrombin. 2ml of fibrinogen and 300 microliters of thrombin were simultaneously dispensed into each mold. In filling the mold with the reagentThereafter, they were returned to the-80 ℃ cooler, maintained for at least 2 hours, and then placed in a freeze-dryer. The dressing was then lyophilized as described. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						Copper (Cu)	6/6	3.7	0.5	153	37.3

Example 49

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. Cooling the mold from-80 deg.CThe cooler was removed and placed on a steel freeze dryer shelf set at-50 ℃. The mold was held on the rack for 30 minutes, allowing the mold temperature to equilibrate to-50 ℃. After 30 minutes, the repeat pipettor was filled with fibrinogen and the second repeat pipettor was filled with thrombin. 2ml of fibrinogen and 300 microliters of thrombin were simultaneously dispensed into each mold. After filling the molds with the reagents, they were kept inside a freeze-dryer at-50 ℃ for 30 minutes. They were then returned to a-80 ℃ cooler for 24 hours before freeze-drying. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						Steel	5/5	3.4	0.5	106	11

And (3) knotting:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						Perforated aluminium	6/6	3.8	0.4	163	31.5
Aluminium	5/5	3.4	0.5
						Copper (Cu)	6/6	3.7	0.5	153	37.3
Steel	5/5	3.4	0.5	106	11

And (4) conclusion:

dressings produced on metal plates that allow rapid freezing and rapid heat transfer passed all experimental criteria. It is expected that other metals or similar materials that can transfer heat at corresponding rates will produce similar results. Example 50

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						Dry ice	6/6	3.7	0.5	153	37.3

Example 51

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. A 18 gauge needle was used to punch 3 holes in the top of the mold. One hole was used for injection of fibrinogen, a second for injection of thrombin and a third hole was used as a vent to release air displaced from the interior of the mold. The pipettor was then filled with fibrinogen and the second pipettor was filled with thrombin. 0.78ml of fibrinogen and 0.17ml of thrombin were simultaneously injected into each mold by these pipettors. After filling, the mold was placed on top of a liquid nitrogen bath for 30 seconds, then returned to a-80 ℃ cooler for at least 2 hours before being placed in a freeze-dryer. They were then lyophilized as described below and performance tested using EVPA and adhesion tests as described. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion
				Dry ice and liquid nitrogen	5/5	3.4	0.5

Example 52

The gasket material was placed in each 1.5X1.5cm PVC mold. 15 microliters of 2% sucrose was pipetted onto the top of each of the four corners of the liner material. The mold was then placed in a-80 ℃ cooler for at least 60 minutes.

Vials containing 3 grams of fibrinogen (Sigma Lot # F-3879) were removed from the-20 ℃ cooler and placed at 4 ℃ for 18 hours. The bottle was then removed from the cooler and allowed to warm to room temperature for 60 minutes. 60ml of water at 37 ℃ were added to the bottle and mixed for 15 minutes at 37 ℃. After being in solution, fibrinogen was dialyzed against IFB. At the end of four hours, HSA was added to a concentration of 80mg/g total protein, and Tween80 (animal derived) was added to a concentration of 15mg/g total protein. The final pH of fibrinogen was 7.4. + -. 0.1. The fibrinogen concentration was adjusted to 37.5mg/ml using CFB. After preparation, fibrinogen was placed on ice prior to use.

Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. The thrombin concentration was adjusted so as to deliver 0.1 units/mg of fibrinogen (when mixed), which corresponds to 25 units/ml of thrombin prior to mixing. After preparation, thrombin was placed on ice prior to use.

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. A 3ml syringe fitted with an 18 gauge needle was filled with 2ml of fibrinogen and a 1ml second syringe fitted with a22 gauge needle was filled with 0.3ml of thrombin. The contents of both syringes were dispensed simultaneously into each mold. After filling, the mold was placed on top of the liquid nitrogen for 30 seconds, then returned to the-80 ℃ cooler for at least 2 hours before being placed in the freeze-dryer. They were then lyophilized as described. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion
				Liquid nitrogen	5/5	3.6	0.5

Example 53

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the-80 ℃ cooler and placed on an aluminum support in a small nitrogen freezing tunnel. Liquid nitrogen is pumped into the front of the channel where it then changes from liquid to gas. This change in state and pressure as it enters the passageway pushes the gas up obliquely and allows it to flow through the die and out the passageway. The channels and molds were cooled with liquid nitrogen gas for 5 minutes prior to addition of fibrinogen or thrombin.

After 5 minutes, the repeat pipettor was filled with fibrinogen and the second repeat pipettor was filled with thrombin. The aluminum support supporting the mold is slid out of the freezing tunnel. 2ml of fibrinogen and 300. mu.l of thrombin were simultaneously addedIs dispensed into each mold. After filling the molds with reagents, they are returned to the freezing channels. The liquid nitrogen was then turned on and allowed to start for 3 minutes. The dressing was then returned to the-80 ℃ cooler for at least 2 hours before freeze-drying. As a result:

group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						Liquid nitrogen vapour	3/6	2.8	0.98	93	48

Example 54

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the 80 ℃ cooler and placed on a steel freeze dryer shelf. Shelf temperatures were as follows: -5 ℃, -10 ℃, -15 ℃, -20 ℃, -25 ℃, -30 ℃, -40 ℃ and-50 ℃. At each temperature, the mold was placed on a shelf for 30 minutes to equilibrate. After 30 minutes, the repeat pipettor was filled with fibrinogen and the second repeat pipettor was filled with thrombin. 2ml of fibrinogen and 300 microliters of thrombin were simultaneously dispensed into each mold. After filling the molds with the reagents, they were kept inside the freeze-dryer at the set temperature for 30 minutes. They were then returned to a-80 ℃ cooler for 24 hours before freeze-drying. As a result:

group siloxane Coolant (. degree. C.)	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						-5	5/5	0.3	0.4	15.2	22.0
-10	5/5	2.2	0.8	80.0	20.5
						-15	5/5	1.4	0.5	62.0	13.4
-20	3/5	2.0	0.7	72.0	18.2
						-25	5/5	2.5	1.5	82	41.0
-30	5/5	2.8	0.4	88	22.4
						-40	4/5	2.8	1.1	108	54.8
-50	5/5	3.4	0.5	106	11.0

Example 55

The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The mold was removed from the 80 ℃ cooler and placed on the shelf of a steel freeze-dryer. Shelf temperatures were as follows: -10 ℃, -20 ℃, -30 ℃ and-40 ℃. At each temperature, the mold was placed on a shelf for 30 minutes to equilibrate. A 3ml syringe fitted with an 18 gauge needle was filled with 2ml of fibrinogen and a 1ml second syringe fitted with a22 gauge needle was filled with 0.3ml of thrombin. The contents of both syringes were dispensed simultaneously into each mold. After filling the molds with the reagents, they were kept inside the freeze-dryer at the set temperature for 30 minutes. They were then returned to the-80 ℃ cooler for at least 2 hours before freeze-drying.

In addition, a set of dressings was produced by placing on an aluminum plate placed either on top of dry ice alone or then on liquid nitrogen for another 30 seconds. A 3ml syringe fitted with an 18 gauge needle was filled with 2ml of fibrinogen and a 1ml second syringe fitted with a22 gauge needle was filled with 0.3ml of thrombin. The contents of both syringes were dispensed simultaneously into each mold. After filling the molds with the reagents, they were kept on dry ice for 5 minutes or placed on liquid nitrogen for 30 seconds. They were then returned to the-80 ℃ cooler for at least 2 hours before freeze-drying.

Group siloxane Coolant (. degree. C.)	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						-10	2/5	0.1	0.2	6.0	12.5
-20	2/4	0.0	0.0	0	0
						-30	4/5	1.3	1.2	68	39.2
-40	3/5	1.5	1.2	74	77.8
						Dry ice (-78 ℃ C.)	4/5	1.8	1.3	50	47.1
Dry ice (-78 ℃ C.) and liquid nitrogen (-196 ℃ C.)	5/5	2.8	0.8	126	39.6

Small knot

Group of	EVPA # PASS/COUNT	Peel test adhesion	Standard deviation of adhesion	Weight maintenance (average) (g)	Standard deviation of weight retention
						Dry ice (-78 ℃ C.)	6/6	3.7	0.5	153	37.3
Dry ice (-78 ℃ C.)	4/5	1.8	1.3	50	47.1
						Dry ice (-78 ℃ C.) and liquid nitrogen (-196 ℃ C.)	5/5	3.4	0.5
Dry ice (-78 ℃ C.) and liquid nitrogen (-196 ℃ C.)	5/5	2.8	0.8	126	39.6
						Liquid nitrogen (-196 ℃ C.)	5/5	3.6	0.5
Liquid nitrogen vapor (-196 ℃ C.)	3/6	2.8	0.98	93	48
						Silicone coolant	5/5	0.3	0.4	15.2	22.0

-5℃
						Silicone Coolant-10 deg.C	5/5	2.2	0.8	80.0	20.5
Silicone Coolant-10 deg.C	2/5	0.1	0.2	6.0	12.5
						Silicone Coolant-15 deg.C	5/5	1.4	0.5	62.0	13.4
Silicone Coolant-20 deg.C	3/5	2.0	0.7	72.0	18.2
						Silicone Coolant-20 deg.C	2/4	0.0	0.0	0	0
Silicone Coolant-25 deg.C	5/5	2.5	1.5	82	41.0
						Silicone Coolant-30 deg.C	5/5	2.8	0.4	88	22.4
Silicone Coolant-30 deg.C	4/5	1.3	1.2	68	39.2
						Silicone Coolant-40 deg.C	4/5	2.8	1.1	108	54.8
Silicone Coolant-40 deg.C	3/5	1.5	1.2	74	77.8
						Silicone Coolant-50 deg.C	5/5	3.4	0.5	106	11.0

Example 56

The liner material was cut and placed in a 2.4X2.4cm per PETG die. 25 microliters of 2% sucrose was pipetted on top of each of the four corners of the liner material. After completion, the mold was placed in a-80 ℃ cooler for at least 60 minutes. Fibrinogen was formulated in CFB (ERL, lot 3150). The fibrinogen concentration was adjusted to 37.5mg/ml using CFB. The final pH of fibrinogen was 7.4. + -. 0.1. After preparation, fibrinogen was placed on ice prior to use. Thrombin was formulated in CTB. The final pH of thrombin was 7.4. + -. 0.1. The thrombin concentration was adjusted with CTB to deliver 0.1 units/mg fibrinogen or 25 units/ml thrombin. The temperature of fibrinogen and thrombin prior to dispensing was 4 ℃. + -. 2 ℃. The three molds were then removed from the-80 ℃ cooler and placed on an aluminum plate pre-cooled on dry ice. The repeat pipettor was filled with fibrinogen and the second repeat pipettor was filled with thrombin. 2ml of fibrinogen and 300 microliters of thrombin were simultaneously dispensed into each mold. After filling the moulds with the reagents, they are returned to the-80 ℃ cooler for at least 2 hoursAnd then placed in a freeze dryer. This is the "control" procedure used for the experiment. The remaining mold was removed from-80 ℃ and placed on a laboratory bench to allow equilibration to room temperature (25 ℃. + -. 5 ℃). The repeat pipettor was filled with fibrinogen and the second repeat pipettor was filled with thrombin. 2ml of fibrinogen and 300 microliters of thrombin were dispensed into the mold simultaneously. The mold was allowed to stand at room temperature for the following times (1, 5, 10 and 15 minutes). At the end of each time point, the mold was placed on an aluminum plate, previously cooled on dry ice, for 5 minutes. At the end of 5 minutes, the mold was returned to the-80 ℃ cooler for at least 2 hours and then placed in a freeze dryer. They were then lyophilized and tested for their performance and biochemical properties as described. As a result:conclusion

A fully functional dressing was produced using a control method containing 31% free alpha chain and no gamma-gamma dimer similar results were seen when the method was modified to allow mixed fibrinogen and thrombin to sit at room temperature for 1 minute. Their performance was still acceptable when the reactants were mixed and held for 5 minutes, although with some reduction, a 9% level of γ - γ dimer was detected in these dressings. Further increasing this time to 10 minutes caused an unacceptable loss of activity in the EVPA assay and increased amounts of free alpha chain and high levels of gamma-gamma dimer (39%). Increasing the hold time to 15 minutes causes a loss of both tack and the ability to hold any weight.

EVPA performance test equipment and spare parts: on-line high pressure sensor (Ashcroft Duralife or equivalent), peristaltic pump (Pharmacia Biotech, Model P-I or equivalent), voltmeter (Crafsman Professional Model 82324 or equivalent), computer equipped with software for recording pressure or voltage information, Tygon tube with attachment (various sizes), water bath (Baxter Durabath or equivalent), pre-set 37 ℃. thermo-insulated chamber (VWR, Model 1400G or equivalent), pre-set 37 ℃. for monitoring waterBath and oven temperature thermometers-various forceps, hemostats, and scissors-10 cc. and 20cc. syringes, drilled approximately 0.6cm hole in the center and smaller drilled holes through both the syringe and the piston. This hole drilled into the end of the syringe is used to keep the piston retracted and secured. O-rings (No. 10 and 13), plastic sleeves (approximately 3.5cm in length) to fit 10cc and 20cc syringes, P-1000 pepeltman with ends, sphygmomanometer with newborn-sized cuff and air bladder, Programmable Logic Controller (PLC) to control the pump to maintain the desired pressure profile (optionally, manual control may be used if desired). 1. Materials and chemicals porcine descending aorta (Pel-Freez Biologicals, Catalog #59402-2 or equivalent), cyanoacrylate glue (Vetbond, 3M or equivalent), 18 gauge needle, 0.9% saline, maintenance at 37 deg.C, Red food dye blood vessel perforator, 2.8mm or other size plastic roll 2. cleaning and storage of arteries 1. storage of arteries at-20 deg.C before use 2. in H₂The arteries were thawed in an O bath at 37 ℃. 3. Fat and connective tissue are cleaned from the outer surface of the artery. 4. The artery was cut into 5cm sections. 5. The arteries can be refrozen to-20 ℃ and stored for future use. 3. Arterial preparation for test 1. the artery was inverted out so that the smooth interior wall faced outward. 2. A 13O-ring was deployed on a 20cc syringe, or a 10O-ring was deployed on a 10cc syringe, with a hole drilled on one side of approximately 0.6cm (0.25 in). 3. The artery is pulled onto the syringe taking care not to tear the artery or create a too loose fit. The artery should fit snugly over the syringe. Another O-ring of the same size is slid onto the bottom of the syringe. 4. Carefully pull the two O-rings over the ends of the artery. The distance of the O-rings should be at least 3.5 cm. 5. The surface of the artery is gently shaved using the blades of some surgical scissors to roughen the surface of the artery. 6. An 18 gauge needle is used to pierce a hole through the artery at the location of the hole in the syringe barrel (see description above) 7. the tip of the biopsy punch is inserted through the hole in the artery. The piston of the perforator is depressed to create a hollow hole in the artery. Repeated several times to ensure that the pores are hollow and free of connective tissue. 8. The foramen was repaired with a collateral artery. Usually, this is done by reacting from latexThe glove was cut to remove the patch and adhered to the hole with cyanoacrylate glue. The glue is allowed to cure for at least 10 minutes. Placing the artery in a warm, humidified container and in a warm-keeping chamber. The artery was allowed to warm for at least 30 minutes. 4. Solution and equipment preparation 1. visual inspection of the water bath and holding chamber was maintained at 29-33 ℃. 2. Ensure that there is enough 0.9% saline in the container of the pump to complete the one day test. More brine is added if needed. 3. 0.9% saline and 0.9% saline to which a few drops of red food coloring were added to the container of the water bath so that the solution was warmed before conducting the test. 4. A container for warming the artery was prepared in a greenhouse by lining with Kim Wipes and adding a small amount of water to keep the artery moist. 5. The tube is checked for bubbles. If air bubbles occur, the pump is turned on and 0.9% saline is allowed to flow until all air bubbles are removed. 5. Application of the dressing 1. open the hemostatic dressing pouch and remove the hemostatic dressing 2. place the hemostatic dressing on the hole in the artery with the mesh pad side up. 3. The hemostatic dressing was slowly wetted with saline in an amount appropriate to the article to be tested. Description of the drawings: standard (13-15 mg/cm)²Fibrinogen) 2.4x2.4cm hemostatic dressing should be wetted with 800 μ l saline or other blood substitute. The amount of brine used may be adjusted as required by the particular experiment being conducted; however, any changes should be recorded in the collected data table. Description of the drawings: the hemostatic dressing is moistened by dropping 0.9% saline or other blood substitute warmed to 29-33 c, and any significant difference in the wettability from the positive control in keeping saline flowing from the edges should be recorded on the data collection table. Gently place the plastic wrap over the hemostatic dressing, taking care to lay it flat between the O-rings. Gently press to secure in place. 5. The artery and hemostatic dressing 6 are wrapped with a plastic wrap and wrapped with a blood pressure cuff, taking care to keep the balloon adjacent to the hemostatic dressing. 7. The balloon was inflated to 100-. The pressure was maintained for 5 minutes. Description of the drawings: the time and pressure can vary depending on the requirements of the experiment and any change from standard conditions should be recorded on the collected data table. 8.After polymerization, the artery was carefully unrolled and the condition of the hemostatic dressing was recorded. Any change from the positive control should be recorded on the data collection sheet.

Exclusion criteria: the mesh pad must remain over the hole in the artery. If it moves during polymerization and does not completely cover the hole, the hemostatic dressing must be removed.

Test method 1. schematic of test apparatus assembly the assembly of the test apparatus is shown in figure 2. Some additional not shown means may be utilized to read (manometer) or control the pressure inside the system 2. the device and artery assembly fills the artery and syringe with 0.9% red saline warmed to 37℃, taking care to minimize the amount of air bubbles in the syringe and artery. Filling the artery with the highest opening may help reduce bubbles. The artery and syringe were connected to the test apparatus to ensure that the bubbles in the tube were as small as possible. The peristaltic pump should be calibrated to deliver approximately 3 ml/min. If available, the PLC should operate according to a predetermined pressure range and hold time determined for the appropriate condition of the article to be tested. If manually controlled, the pressure/time profile is followed by manually turning the pump on and off while referencing the system pressure as read by one or more pressure reading components of the system. After the test was completed, the hemostatic dressing was subjectively evaluated for adhesion to the artery and formation of emboli in the arterial ostia. Any change from the positive control should be recorded on the data collection sheet. Success criteria

A hemostatic dressing capable of withstanding pressure for 3 minutes is considered to pass the test. When the hemostatic dressing successfully passed the test, data collection should be stopped immediately so that the natural reduction in pressure in the artery that occurs at the end of the test is not included in the figure. If the operator fails to stop data collection, these points can be deleted from the data file to avoid confounding the natural pressure drop that occurs after the test with actual dressing failure. The predetermined criteria must be met throughout the trial from the application of the hemostatic dressing to completion. The maximum pressure reached should be recorded on the data collection table. Description of the drawings: a typical challenge in one step is 250mmHg, 3 minutes, but may vary depending on the article to be tested. Changes to the standard method should be recorded on the collected data table. Failure criteria

Hemostatic dressings that begin to leak saline at any point during the test are considered to fail the test. Description of the drawings: failure of the construct due to arterial dilation is negligible and the trial or restart trial is continued (as long as the total trial time does not fall outside the determined limits).

When the leak occurred, the pressure should be reduced to-20 mmHg, after which the data collection should be stopped so that the failure is easily observed on the graph. The pressure at which the leak occurred should be recorded on the data collection table. If data collection is stopped due to equipment failure in the middle of the experiment, data can be collected manually at 5 second intervals until the end of the experiment or the hemostatic dressing fails, whichever occurs first. Data points should be recorded on the back of the collected data sheet, marked explicitly, and entered into the data sheet manually. Exclusion criteria

If the total test time exceeds the maximum time allowed by the method, the results obtained must be excluded, whatever the cause. If there is a leak due to the collateral artery not being fixed by repair or finger pressure, the results obtained must be excluded. If the test fails due to O-ring leakage, the results obtained must be excluded. If the mesh pad does not completely cover the hole in the artery, the results obtained must be excluded. Adhesion Performance test 1. Equipment and spare parts

Preparation of hemostat, porcine artery and hemostatic dressing (usually after completion of EVPA test, although it is not necessary for adhesion test to be performed) i

After the dressing was applied without completing the EVPA test, the dressing was ready for adhesion testing and weight limit testing (if applicable). After application of the dressing and subsequent EVPA analysis, the arterial and syringe system was then slowly disconnected from the pump so that the solution was not sprayed around. The warm red saline solution from the EVPA test was retained in the syringe until the adhesion test and weight limit test (if applicable) were completed.

Performance of adhesion test 1. after preparation of the artery and dressing (with or without EVPA analysis), one corner of the mesh was gently lifted and a hemostat of known quality was attached to the corner. Description of the drawings: if FD forms a channel leak during EVPA test performance, adhesion tests are performed on the opposite side of the hemostatic dressing in order to obtain a more accurate assessment of total adhesion. Let go of the hemostat gently, taking care not to drop or twist the hemostat. The syringe is turned so that the hemostat is near the top and the hemostat is allowed to peel the dressing as far as the dressing will allow. This is typically done in 10 seconds. After the hemostat stopped peeling the dressing, the adhesion of the bandage was scored according to the following criteria:

dressing performance scoring	Amount of tack
		4	90+％
3	75-90％
		2	50-75％
1	-50％
		0.5	Hemostatic forceps for keeping only embolism
0	Without sticking

Exclusion criteria

The mesh pad must remain over the hole in the artery. If it moves during polymerization and does not completely cover the hole, the hemostatic dressing must be removed. Success criteria

A dressing with an adhesion score of 3 is considered to pass this test. Failure criteria

If the dressing did not adhere to the artery after application or before the EVPA test was performed, it scored 0 and the adhesion test failed. If the dressing received a score of 2 or less, the dressing was considered to have failed the adhesion test. Weight retention Performance test

After the initial scoring of the "tack test," the hemostat may then be incrementally weighted until the mesh pad is completely pulled from the artery. The maximum weight held by the dressing is then recorded as a measure of the weight that the dressing can hold attached to the artery. Moisture test

Moisture tests were performed using a Brinkman Metrohm Moisture Analyzer System. The system contained the following individual components, 774 oven sample handler, 774SC controller, 836 titrrando, 5ml and 50ml 800 Dosino Units and 801 stirrer. The system was connected to a computer using the Brinkman Tiamo software for data collection, analysis and storage. The moisture system was assembled and run according to the manufacturer's recommendations and instructions to measure the moisture content of the lyophilized sample using the karl fischer method.

All parts were opened and brought to operating temperature before use. Lactose and water were run as standard samples and used to calibrate the instrument. After the machine was successfully calibrated, the samples were prepared as follows. Pieces of dressing weighing at least 30mg were placed in vials and covered with lids. The vials are placed 774 in the oven sample handler in the numbered order, and an empty capped vial is placed in the ambient space (conditioning space). The machine was then run to test the moisture content (residual moisture) in the control and sample. SDS-PAGE gel electrophoresis

Each dressing was cut into quarters, approximately 50mg each, and one portion was placed in a 15mL conical tube. To generate a control (i.e., time 0), 1.0mL of Okuda dispensing Solution (10M urea, 0.1% sodium lauryl sulfate, 0.1% beta-mercaptoethanol) was added. For the remaining 3 pieces, 80 μ L of 0.9% saline was added to wet the dressing. The pieces were then incubated at 37 ℃ for 2, 5, and 10 minutes or the desired time. To stop the reaction at the desired time, 1.0mL of Okuda Dissolvangsolution was added. The samples were then incubated overnight at room temperature and then at 70 ℃ for 30 minutes.

To prepare the sample for loading on the gel, the sample previously dissolved in the Okuda dispensing Solution was added to the sample buffer so that a 20. mu.L aliquot contained 10. mu.g. Then 1. mu.L of 0.1M dithiothreitol was added to each sample. Mu.l of each diluted sample was then loaded on 10 wells of 1.0mm thick 8% Tris-glycine gel (Invitrogen). The gel was then run at 140V until the dye front reached the end of the gel. It was then removed and the Coomassie blue colorant (in ddH) on a vibrating platform₂50% v/v methanol in O, 0.25% w/v Coomassie Brilliant blue, 10% w/v acetic acid) for at least 1 hour. The gel was then transferred to a destaining solution (25% methanol, 10% acetic acid, 65% ddH) on a shaking platform₂O) until the background is almost colorless.

After decolourisation, the gel was scanned and the gamma-gamma dimer bands and the a alpha and B beta bands were analysed by Scion density test software in order to determine the amount of conversion that occurred.

Claims

1. A method of producing a solid dressing for treating wounded tissue in a mammal, comprising: (a) forming a liquid aqueous mixture of the fibrinogen component and the fibrinogen activator at a temperature sufficiently low to inhibit activation of the fibrinogen component by the fibrinogen activator; (b) reducing the temperature of the aqueous mixture to form a frozen aqueous mixture; and (c) reducing the moisture content of the frozen aqueous mixture to produce a solid dressing having a haemostatic layer consisting essentially of the fibrinogen component and the fibrinogen activator.

2. The method of claim 1, wherein the solid dressing further comprises at least one support layer.

3. The method of claim 2, wherein the support layer comprises a backing material.

4. The method of claim 3, wherein the support layer comprises an internal support material.

5. The method of claim 3, wherein the support layer comprises a resorbable material.

6. The method of claim 3, wherein the support layer comprises a non-resorbable material.

7. The method of claim 6 wherein said non-resorbable material is selected from the group consisting of silicone polymers, paper, gauze, plastics, metals, and latex.

8. The method of claim 3, wherein the solid dressing further comprises at least one physiologically acceptable sealant between the haemostatic layer and the support layer.

9. The method of claim 5, wherein the resorbable material is selected from the group consisting of proteinaceous materials and carbohydrate materials.

10. The method of claim 9, wherein the proteinaceous material is at least one substance selected from the group consisting of keratin, silk, fibrin, collagen, and gelatin.

11. The method of claim 9, wherein the carbohydrate material is selected from the group consisting of alginic acid and salts thereof, chitin, chitosan, cellulose, N-acetylglucosamine, proteoglycans, hyaluronidase, hyaluronic acid, glycolic acid polymers, lactic acid polymers, glycolic acid/lactic acid copolymers, and mixtures of two or more thereof.

12. The method of claim 1, wherein the haemostatic layer further comprises a fibrin cross-linking agent and/or a source of calcium ions.

13. The method of claim 1, wherein the haemostat layer further comprises one or more of: at least one filler, at least one solubilizer, at least one blowing agent, and at least one release agent.

14. The method of claim 13, wherein the filler is selected from the group consisting of sucrose, lactose, maltose, keratin, silk, fibrin, collagen, gelatin, albumin, polysorbate, chitin, chitosan, alginic acid and salts thereof, cellulose, proteoglycans, hyaluronidase, hyaluronic acid, glycolic acid polymers, lactic acid polymers, glycolic acid/lactic acid copolymers, and mixtures of two or more thereof.

15. The method of claim 13, wherein the solubilizing agent is selected from the group consisting of sucrose, lactose, maltose, dextrose, mannose, trehalose, mannitol, sorbitol, albumin, hyaluronidase, hyaluronic acid, sorbate, polysorbate, and mixtures of two or more thereof.

16. The method of claim 13, wherein the release agent is selected from the group consisting of gelatin, hyaluronidase, hyaluronic acid, mannitol, sorbitol, polysorbate, sorbitan, lactose, maltose, trehalose, sorbate, glucose, and mixtures of two or more thereof.

17. The method of claim 13, wherein the blowing agent is selected from the group consisting of: sodium bicarbonate/citric acid, sodium bicarbonate/acetic acid, calcium carbonate/citric acid, and calcium carbonate/acetic acid, and carbohydrate-entrained pressurized inert gas.

18. The method of claim 1, wherein the haemostatic layer further comprises at least one therapeutic supplement selected from the group consisting of antibiotics, anticoagulants, steroids, cardiovascular drugs, growth factors, polyclonal and monoclonal antibodies, chemotactic agents, anesthetic agents, antiproliferative/antineoplastic agents, antiviral agents, cytokines, colony stimulating factors, antifungal agents, antiparasitic agents, anti-inflammatory agents, antiseptics, hormones, vitamins, glycoproteins, fibronectin, peptides, proteins, carbohydrates, proteoglycans, anti-angiogenin, antigens, nucleosides, lipids, liposomes, fibrinolysis inhibitors, and gene therapy agents.

19. The method of claim 18, wherein the therapeutic supplement is present in an amount equal to or greater than its solubility limit in fibrin.

20. The method of claim 2, wherein the haemostat layer further comprises at least one sealant in an amount effective to improve adhesion of the haemostat layer to the support layer.

21. The method of claim 20, wherein the sealant is selected from the group consisting of sucrose, mannitol, sorbitol, gelatin, hyaluronidase, hyaluronic acid, maltose, povidone, chitosan, and carboxymethylcellulose.

22. The method of claim 1, wherein the entire layer of the haemostat layer is substantially homogeneous.

23. The method of claim 1, wherein the haemostat layer is monolithic.

24. The method of claim 1, wherein the frozen aqueous mixture is lyophilized in (c).

25. The method of claim 1, wherein the moisture content is a minimum of 6%.

26. The method of claim 1, wherein the moisture content is less than 6%.

27. The method of claim 1, wherein the fibrinogen component is mammalian fibrinogen.

28. The method of claim 27, wherein said mammalian fibrinogen is selected from the group consisting of bovine fibrinogen, porcine fibrinogen, ovine fibrinogen, equine fibrinogen, caprine fibrinogen, feline fibrinogen, canine fibrinogen, murine fibrinogen, and human fibrinogen.

29. The method of claim 1, wherein the fibrinogen component is selected from the group consisting of human fibrinogen, human fibrin I, human fibrin II, human fibrinogen α chain, human fibrinogen β chain, human fibrinogen γ chain, and mixtures of two or more thereof.

30. The method of claim 28 or 29, wherein the fibrinogen is selected from the group consisting of recombinantly produced fibrinogen and transgenic fibrinogen.

31. The method of claim 1, wherein the fibrinogen activator is selected from the group consisting of thrombin, prothrombin, snake venom, and mixtures of any two or more thereof.

32. The method of claim 31, wherein the thrombin is mammalian thrombin.

33. The method of claim 32, wherein the mammalian thrombin is selected from the group consisting of bovine thrombin, porcine thrombin, ovine thrombin, equine thrombin, caprine thrombin, feline thrombin, canine thrombin, murine thrombin and human thrombin.

34. The method of claim 32, wherein the thrombin is selected from the group consisting of recombinantly produced thrombin and transgenic thrombin.

35. A solid dressing for treating wounded tissue in a mammal prepared by the process of claim 1.

36. A frozen aqueous mixture prepared according to (a) and (b) of claim 1.

37. An aqueous mixture of liquids prepared according to (a) in claim 1.

38. A solid dressing for treating wounded tissue in a mammal prepared by the process of claim 1, further comprising less than 57% free fibrinogen α -chain.

39. A solid dressing for treating wounded tissue in a mammal prepared by the process of claim 1, further comprising substantially no free fibrinogen α -chain.

40. A solid dressing for treating wounded tissue in a mammal prepared by the process of claim 1, further comprising substantially no fibrin Ia.

41. A solid dressing for treating wounded tissue in a mammal prepared by the process of claim 1, further comprising substantially no fibrinogen chain gamma dimers.

42. A solid dressing for treating wounded tissue in a mammal prepared by the process of claim 1, further comprising less than 9% fibrinogen chain gamma dimer.

43. The method of claim 2, wherein the support layer comprises a front side support material.

44. The method of claim 1, wherein the temperature is reduced to-10 ℃ to-196 ℃.

45. The method of claim 1, wherein the liquid aqueous mixture is frozen in either a horizontal or vertical direction while reducing the temperature.

46. A solid dressing for treating wounded tissue in a mammal prepared by the process of claim 1, further comprising up to 9% fibrinogen chain gamma dimer and up to 57% free fibrinogen alpha chain.