Microstructured Hemostatic Agent

Abstract

The microstructured hemostat comprises a hierarchical microstructured surface (300) composed of multiple layers of microstructures (302, 306, 308, 310), each characterized by one or more length scales. The microstructured hemostat of the present invention can shorten blood clotting time, control clot morphology, and provide a novel diagnostic platform for assessing clotting function from a morphological perspective.

Description

Technical Field

[0001] The present disclosure generally relates to devices and methods useful as diagnostic surfaces useful for perioperative arrest of bleeding, correction of coagulation defects by promoting a defined blood clot morphology, and evaluation of coagulation morphology. Such devices and methods are useful for surgical procedures, prevention of postoperative adhesions, maintenance of patent lumens, and quantification of coagulation lesions. Further, such hemostatic devices are useful for local recovery of pharmaceutically induced coagulation defects. More particularly, the present disclosure relates to multifunctional hierarchical microstructures and three-dimensional immobilization interface domain structures that result in super slippery and super adhesive devices.

Background Art

[0002] Changes in the coagulation pathway play an important role in the outcome of blood clot propagation. The structure-function relationship of the developing fibrin mass is known to be affected by many factors such as environment, treatment, and disease as compared to normal blood clot growth. In some cases, environmental factors can compensate for deficiencies due to disease factors.

[0003] The primary microstructure of the fibrin mass consists of an irregular network of intertwined and branched fibrin fibers. Thinner fibers are associated with networks with an increased number of branch points and create a denser and less permeable blood clot with a known association with thrombotic embolism disease. This observation suggests that microstructured surfaces can play a role in inducing the structure of the blood clot by adapting the hierarchical structure of the hemostatic agent / implant to the desired branching ratio of the fibrin fibers.

[0004] Blood clots with altered fibrin microstructure exhibit different sensitivities to fibrinolysis, and the permeability of the blood clot is the rate-limiting factor for the activity of plasmin, a fibrin network degrading enzyme.

[0005] A properly patterned arterial stent can, when a blood clot is formed, change the permeability of the blood clot, thereby assisting or interfering with the ability of tissue plasmin activator and / or urokinase plasmin activator to move through the three-dimensional fibrin network and activate prothrombin plasminogen into fibrinolytic plasmin.

[0006] Blood clotting is known to involve the formation of solid and liquid fractal and hierarchical domains. These domains have been found to correspond to differences in surface energy that occur self-similarly at many size scales. Furthermore, it has unexpectedly been found that a solid surface patterned with hierarchically arranged alternating high-energy and low-energy regions can significantly extend and / or shorten the clotting time by predicting the natural clot microstructure.

[0007] In that case, a microstructured surface is required to overcome the problem of rapid cessation of blood flow due to a defect in the living tissue.

[0008] Furthermore, there is an increasing need to provide functional biomarkers for "health indicators" of normal clotting that can monitor the effects of therapeutic procedures and diseases on the quality and fate of blood clots. SUMMARY OF THE INVENTION

[0009] In one embodiment, the present disclosure provides a device for promoting blood clotting. The device can include a hierarchically arranged microstructured surface configured to have a fractal dimension between 2 and 3, the microstructured surface being configured to promote thrombus formation when in contact with blood. In some embodiments, the device can include a fractal dimension between 2.06 and 2.08. In still other embodiments, the device can include a fractal dimension of 2.07.

[0010] In one embodiment, the device can include a hierarchically arranged microstructured surface that includes at least a first surface texture and a second surface texture. Due to this hierarchical arrangement, the second surface texture can be disposed on the first surface texture. Further, at least two surface textures can be configured to generate a plurality of spatially distributed surface energy gradients.

[0011] In one embodiment, the hierarchically arranged microstructured surface further includes a third surface texture, and this third surface texture is hierarchically arranged on the second surface texture. Further, the first surface texture is arranged in a triangular lattice and can include a height of at least 400 microns. The second surface texture can have a diameter of 25 microns and a height of 45 microns. The third surface texture can have a diameter of 5 microns and a height of 15 microns. In one embodiment, the second surface texture and the third surface texture may also be arranged in a triangular lattice, similar to the first surface texture, respectively.

[0012] In one embodiment, the third surface texture may further include a vertical surface with longitudinal grooves, and each longitudinal groove can have a width of 5 microns.

[0013] In some embodiments, the hierarchically arranged microstructured surface is configured to have a shortened activated partial thromboplastin time compared to a surface lacking a hierarchical microstructural arrangement.

[0014] In some embodiments, a device for separating blood components can include hierarchically arranged microstructured surfaces, which can include a first distinct surface and a second distinct surface. The first and second distinct surfaces may each have a center, and the center of the first distinct surface may be located at least 50 microns from the center of the second distinct surface. The first distinct surface can be configured to localize a first blood component that substantially contains solids, and the second distinct surface can be configured to localize a second blood component that substantially contains fluids.

[0015] In one embodiment, the first distinct surface can include at least two hierarchically arranged microstructured textures. The first distinct surface has a first fractal dimension. The second distinct surface can include at least two hierarchically arranged microstructured textures, and this second distinct surface has a second fractal dimension. In some embodiments, the second distinct surface can be configured to remove the second blood component by capillary action.

[0016] In one embodiment, the first distinct surface and the second distinct surface can cooperate such that the hierarchically arranged microstructured surface can have a shortened activated partial thromboplastin time compared to a surface lacking a hierarchical microstructure arrangement.

[0017] In some embodiments, a device for promoting blood coagulation can include a surface having a hierarchical microstructure and a chemical coagulant. The chemical coagulant may be cross-linked to the microstructure.

[0018] In some embodiments, the chemical coagulant can include Gardenia fruit extract, free amino groups of collagen / gelatin, and / or at least one oxidized polysaccharide. In some embodiments, the surface can include a polymeric substrate containing at least one aldehyde group. BRIEF DESCRIPTION OF THE DRAWINGS

[0019]

Figure 1

Figure 2

Figure 3

Figure 3A

Figure 4

Figure 4A

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0020] Here, embodiments of the present disclosure will be referred to in detail, and one or more of the drawings will be described herein. Each drawing is provided for the purpose of explaining the present disclosure and is not limiting. In fact, it will be apparent to those skilled in the art that various modifications can be made to the teachings of the present disclosure without departing from the scope of the present disclosure. For example, features illustrated or described as part of one embodiment can be used with another embodiment to obtain still another embodiment.

[0021] Accordingly, the present disclosure is intended to cover modifications and variations that fall within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present disclosure are disclosed in the following detailed description or will be apparent from the following detailed description. Those skilled in the art will understand that this discussion is merely illustrative of exemplary embodiments and is not intended to limit the broader aspects of the present disclosure.

[0022] As used herein, the term "microscopic" is understood to include a mechanism of dimensions small enough to require visual aids to the naked eye when viewed from any observation plane to determine its shape. Normal vision is considered to be the case where the smallest recognizable character has an angular height of 5 minutes on the retina. At a typical working distance of 250 mm (about 10 inches), for normal vision, the lateral dimension of this object is 0.36 mm (about 0.0145 inches).

[0023] As used herein, the term "microstructure" is understood to include the configuration of a mechanism where at least two dimensions of the mechanism are microscopic. As an example, the topological cross-sectional view of a mechanism can be microscopic.

[0024] As used herein, the term "dimensional feature" is understood to include a mechanism protruding from the body of a microstructured surface having a specific range of sizes. For example, the dimensional feature of a pillar having a radius of 10 - 20 microns and a height of 30 - 40 microns is distinguished from another dimensional feature of a pillar having a radius of 80 - 100 microns and a height of 80 - 125 microns. Generally, the volume of the dimensional features differs by a factor of 10 or more.

[0025] As used herein, the term "positive feature" is understood to include a feature protruding from the body of a microstructuring forming tool, a microstructuring liner, a microstructuring backing, or a microstructuring pressure-sensitive adhesive layer.

[0026] As used herein, the term "negative mechanism" is understood to include a mechanism that is recessed into the body of a microstructured forming tool, a microstructured liner, a microstructured backing, or a microstructured pressure-sensitive adhesive layer.

[0027] As used herein, the term "suction mode" is understood to include a negative mechanism that can generate capillary action.

[0028] As used herein, the term "embossable" is understood to include the ability of a substrate layer to raise a portion of its surface and lower other portions of its surface, particularly by mechanical means.

[0029] As used herein, the term "substrate layer" is understood to include a smooth and flat planar sheet of a thermoplastic material or polymer, such as can receive a microstructure by embossing, molding, printing, and casting, and the microstructure becomes integral with the substrate layer.

[0030] As used herein, the term "wetting" is understood to include spreading over a surface and bringing the surface into intimate contact with a fluid. An aqueous fluid on a hydrophilic surface can be wetting.

[0031] As used herein, the term "dewetting" is understood to include a fluid that contracts from intimate contact with a surface. An aqueous fluid on a hydrophobic surface can be dewetting.

[0032] As used herein, the term "multifunctional microstructure" is understood to include liquid, solid, and / or gas domains that form on a microstructured surface when the surface contacts a target surface. These domains can reduce the surface energies of the microstructured surface and the target surface at the interface between the two surfaces. The interface can be made adhesive by the formation of a low-energy interface that requires energy to be supplied to the interface to separate the two surfaces. Physical forces involved in domain formation can include van der Waals forces, Casimir forces arising from quantum interactions with the zero-point field, intermolecular forces, London dispersion forces, Debye forces, Keesom forces, and hydrogen bonds. An adhesive microstructure does not refer to any chemically reactive substance that grips a target surface by surface coating.

[0033] As used herein, the term "permanently repositionable multifunctional microstructured surface" is understood to include a repositionable microstructured surface whose adhesion strength to a given target substrate cannot substantially change over time under the application conditions.

[0034] As used herein, the term "temporarily repositionable multifunctional microstructured surface" is understood to include a repositionable microstructured surface that builds adhesion with time, pressure, or temperature. This phenomenon is sometimes referred to as the "suction effect" where the interface between the microstructured adhesive and the target surface gradually thins, increasing the energy of dissociation.

[0035] As used herein, the term "dissociation energy" is understood to include the difference in surface energy between the microstructure and the target surface joined at the interface apart. Dissociation energy is understood to be a quantitative measure of the degree of adhesion within the present disclosure.

[0036] As used herein, the term "target substrate" is understood to include a surface to which a pressure-sensitive microstructured adhesive can be applied for the intended purpose.

[0037] As used herein, the term "exclusion zone" is understood to include the region between a surface and water, and due to the hydrophilicity of the surface, due to either surface texture or chemical properties, water can thermodynamically exclude ions and molecules that are not water.

[0038] As used herein, the term "structured water" is understood to include the state of water in which water molecules in a solution take on a transitional hexagonal shape, which can be enhanced and maintained by the surface energy and surface chemistry between the water and the solid surface.

[0039] As used herein, the term "capillary action" (capillarity, capillary motion, capillary rise, capillary effect, or uptake) is understood to include the ability of a liquid to flow through a narrow space without the aid of an external force such as gravity or even against an external force. As an example, this effect can be seen between the hairs of a paintbrush, in thin tubes, in porous materials such as paper and plaster, in some non-porous materials such as sand and liquefied carbon fibers, or in the uptake of liquid within cells. This is understood to occur due to the intermolecular forces between the liquid and the surrounding solid surface. When the diameter of the tube is small enough, the combination of surface tension and the adhesive force between the liquid and the walls of the container can act to propel the liquid.

[0040] As used herein, the term "pitch" is understood to include the center-to-center distance between two adjacent microstructures.

[0041] Generally, the devices of the present application are hierarchically microstructured, which means that the microstructures can be defined by a set of different structure sizes. For example, the pillars can have a diameter of about 1 - 5 microns and can be placed on top of another set of pillars having a diameter of about 25 - 50 microns. This configuration can also be called a complex pillar (a plurality of hierarchically arranged pillar sets) and can be placed on a capillary bed that includes depressions having capillary function.

[0042] These microstructured hierarchical surfaces can present hydrophobic domains (low surface energy) and hydrophilic domains (high surface energy) in proximity or juxtaposition, and it has been determined that they can self-differentiate a composite liquid interface into hydrophilic and hydrophobic zones. These types of microstructured hierarchical surfaces can create a Wenzel-Cassie zone, where a portion of the zone can be hydrophilic (Wenzel) and another portion can be hydrophobic (Cassie). The Wenzel-Cassie zone can encompass oil-based aqueous solutions, composite fluids containing microparticles and water, and / or air / liquid composite fluids.

[0043] The Wenzel-Cassie zone on the device of the present application can be particularly useful in blood contact applications. The Cassie zone can create a large contact angle between the blood and the device surface, while the Wenzel zone can absorb hydrophilic components such as water. In some embodiments, such as blood contact applications, a droplet of blood contacting the microstructured surface can undergo fluid pinning, and red blood cells and other microparticles can be concentrated in the blood volume and plasma (Cassie zone), and the aqueous component can be absorbed by capillary action (Wenzel zone).

[0044] The result of blood pinning and concentration by capillary action can be to concentrate the blood, make clotting more rapid, and form a high-density morphology. Macroscopically, it can be seen that the microstructured device "sucks in" on the bleeding surface and thus stops blood flow. In some embodiments, the cessation of blood flow can be caused prior to actual clotting. The degree of capillary action can be adjusted according to the bleeding flow rate such that a rapidly bleeding surface can be pinned more aggressively than a less bleeding surface. This can be done.

[0045] In some embodiments, the hemostatic agent of the present disclosure can pin more blood in the center and absorb more plasma around it. Such anisotropy can be achieved by changing the size and / or pitch of the microstructure and by affecting the surface energy of the surface by surface treatment such as plasma activation or coating with an ionic compound. Alternatively, the activated surface may be achieved by the generation of nanostructures of the substrate material. For example, the substrate surface can be abraded, chemically dissolved, or decomposed by some mechanical method including the deposition of nanoparticles.

[0046] The surface energy can also be locally adjusted by embedding microelectrodes and then charged by an external source to reversibly change the surface energy. Electrolytic compounds can be used such that when they conduct blood, the compounds can pass an electric current and accumulate charges on separate surfaces.

[0047] In some embodiments, the size and pitch of the microstructures can be selected to promote coagulation and / or thrombus formation. In one embodiment, coagulation can initiate on the scale of 1 micron. These small-scale thrombi can then be combined on a characteristic scale to form larger thrombi. This progression across characteristic dimensions is characteristic of the coagulation cascade. Coagulation can be enhanced by matching the hierarchical structure of the microstructured surface to the characteristic dimensions of the coagulation cascade. It should be understood that coagulation can be considered a phase transition from an irregular fluid state to a regular fibrous solid state. The pattern of local surface energy can catalyze the phase transition from liquid blood to solid thrombus.

[0048] For the coagulation mechanism altered by a disease or a pharmaceutical, in some embodiments, characteristic coagulation dimensions may change. This morphological change in the coagulation kinetics can be expressed as a branching ratio, where fibers of a certain length and diameter preferentially branch, while other fibers of different lengths and / or diameters may be less likely to branch. When fibers branch, their geometric shape can become fractal. Here, the fractal geometric shape refers to a partial geometric shape between a two-dimensional planar geometric shape and a three-dimensional volume geometric shape. The higher the density of the branching points, the closer the geometric shape is to the two-dimensional plane.

[0049] By recognizing the morphological behavior of coagulation, it may be possible to design better hemostatic agents and hemostatic agents that correct specific coagulation kinetics. Furthermore, an array of microtextured surfaces exposed to blood with specific coagulation kinetics can cause a coagulation phase transition on one patterned surface but not on others. By observing the coagulation time on microstructured surfaces with different fractal dimensions, a quantitative means for diagnosing a patient's coagulation state can be provided.

[0050] Thus, embodiments of the present disclosure can be classified into three categories: 1) hemostatic agents having a normal coagulation-promoting fractal dimension, 2) hemostatic agents that catalyze a damaged coagulation pathway having a different fractal dimension, and 3) diagnostic surfaces that measure the fractal dimension of the coagulation pathway. These embodiments may further be combined with a chemical coagulant for yet other embodiments.

[0051] Embodiments of the present disclosure can be further enhanced by recognizing that the microstructured surface can actively induce the morphology of the resulting thrombus. Thrombi with a low branching ratio are more diffuse, porous, and may be easily dissolved. Thrombi with a high branching ratio are more dense, impermeable to fluid penetration, and may be structurally robust. In embodiments where the microstructured surface is in contact with flowing blood, the microstructured surface resists thrombus formation and a thrombus is formed Even if it is composed of a material that promotes the formation of disseminated thrombosis, in this embodiment, the microstructured surface can be resistant to thrombosis formation, and even if thrombosis formation occurs, it can lead to a configuration in which the thrombosis is easily reabsorbed. For example, the formation of a disseminated fibrin structure can promote endothelialization, which can then become strongly antithrombotic. Endothelial cells are known to release nitric oxide, which can prevent thrombosis formation.

[0052] One embodiment of the present invention is a microstructured surface having a specific branching ratio. Referring to FIG. 1, the hierarchical microstructured surface 100 has a branching ratio defined by a hierarchical microstructure including a large microstructure 102, a medium microstructure 104, and a small microstructure 106. In some embodiments, the individual large microstructures 102 can include a pitch of 1000 microns between centers, the individual medium microstructures 104 can include a pitch of 100 microns between centers, and the individual small microstructures 106 can include a pitch of 10 microns between centers. The small microstructures 106 can be disposed on the upper surface 108 of the medium microstructures 104. The medium microstructures 104 can be disposed on the upper surface 110 of the large microstructures 102. In some embodiments, the reference line 112 can define a branching ratio in which the height 114 of the large microstructure is 10 times the height 116 of the medium microstructure 104, and the height 116 of the medium microstructure 104 is 10 times the height 118 of the small microstructure 108. The reference line 112 can define a fractal dimension of 2.1 = 2 + the ratio of successive lengths, where 2 is the dimension of a surface without microstructure. The fractal dimension of normal coagulation is approximately 2.07.

[0053] In some embodiments, it will be appreciated that the fractal dimension may not depend on the shape of the microstructure. Instead of cylindrical pillars, square pillars, or pillars of any polygon can be used. In embodiments where the angle arranged around the microstructure is larger, the surface energy of the microstructure can be increased. A microstructure having a longitudinal groove or finned side structure can be particularly useful for constructing a Wenzel-Cassie structure. For example, referring to FIG. 2, a Wenzel-Cassie microstructure 200 is shown in a top view and can be defined by a large microstructure 202 with smaller microstructures 204 hierarchically arranged thereon. In some embodiments, the large microstructure 202 can include longitudinal grooves 206, and adjacent large microstructures can have longitudinal grooves that interpenetrate as shown in region 208. Due to the interpenetration of the longitudinal grooves, region 208 can be hydrophilic. Due to the hydrophilicity of this embodiment, the fluid can be drawn below the channel 210 by capillary action. Alternatively, in embodiments having smaller microstructures 204 that are close to each other, for example, arranged at least 2 microns apart, the interpenetration region 208 can be hydrophobic. With this configuration, a blood droplet 212 (shown by a dashed line), which may be composed of plasma and red blood cells, can concentrate red blood cells on the upper surface of the large microstructure 202, and the plasma is drawn into the channel 210, so that blood components such as platelets are concentrated and coagulation defects can be compensated for. 218 can be concentrated, and the plasma is drawn into the channel 210, thus concentrating blood components such as platelets and compensating for coagulation defects.

[0054] In some embodiments, a microstructural hemostatic agent having a Wenzel-Cassie microstructure 200 can actively stop blood flow by pulling the microstructure surface in the direction of the tissue surface when plasma is absorbed from the interface surface. Alternatively, such a pattern can be used to separate blood into a liquid component and a solid component and isolate them from each other.

[0055] The hemostatic agent of the present disclosure can also be used in combination with a chemical coagulant such as a fibrin-based sealant composition, with or without collagen and / or an aldehyde-containing polymer. When the fibrin-based sealant is crosslinked to a microstructured polymer having aldehyde groups, thrombin formation with increased sealant strength can be promoted.

[0056] In some embodiments, the fibrinogen component of the fibrin sealant can be derived from a cryoprecipitate concentrated by ultrafiltration. The fibrinogen component can contain a fibrinogen content of about 1 to 100 mg / ml, particularly 50 to 75 mg / ml. The amount of fibrinogen can depend on the surface energy and surface energy gradient of the microstructure of the hierarchically arranged microstructured hemostatic agent.

[0057] Crosslinking responsible for thrombus formation can be formed by Schiff base formation between the free amino groups of collagen / gelatin and the aldehyde groups in the polymer. In some embodiments, the microstructured polymer can contain a polysaccharide. An example of a polysaccharide suitable for use in one embodiment is polymerized dextran. In another embodiment, oxidized polymerized dextran or xanthan can be used.

[0058] The oxidized polysaccharide included in the present disclosure is preferably oxidized dextran in solution. However, other polysaccharides having appropriate viscosity, molecular weight, and oxidation characteristics can also be used in various embodiments.

[0059] The molecular weight of the oxidized dextran used in the production of the wound dressing is preferably less than 5 million Daltons, more preferably 10,000 to 100,000 Daltons. In some embodiments, the viscosity of an aqueous solution of dextran can be, for example, 0.1 to 1 Pas in a 2% solution (when measured using a Brookfield LVT viscometer operated at 30 cycles).

[0060] The oxidation of dextran is a reaction well-known to those skilled in the art. For example, oxidation can be conveniently obtained by treatment with an aqueous solution of a periodate such as sodium periodate. The purpose of the oxidation can be to cause the formation of reactive dialdehyde residues on the polymer-based microstructured surface.

[0061] The above oxidation procedure may be used, but those skilled in the art should understand that other oxidation methods that result in the formation of dialdehyde residues may also be possible in other embodiments. For example, several embodiments for treatment with periodic acid or lead tetraacetate in an organic solvent such as dimethyl sulfoxide can cause oxidation. After oxidation, the oxidized dextran can be easily purified and separated from low molecular weight reaction components by classical purification methods. Examples for achieving this include, but are not limited to, precipitation (e.g., by addition of acetone, methanol or isopropanol) or dialysis, ultrafiltration, or gel permeation chromatography, followed by lyophilization.

[0062] The crosslinking between collagen or gelatin and oxidized dextran can be achieved in situ by the formation of so-called Schiff base bonds between the free amino groups present on collagen / gelatin (especially on its lysine residues) and the dialdehyde residues on oxidized dextran and fibrinogen. These reactions can be catalyzed by the presence of a high surface energy gradient.

[0063] In some embodiments, this reaction is initiated in the presence of blood which can form an aqueous fibrin sealant medium, and the rate and extent of crosslinking can depend on various parameters. The parameters can include the type of collagen / gelatin, the concentration of oxidized dextran, the degree and molecular weight of dialdehyde substitution, pH, the type of buffer, and / or the presence of electrolytes in the reaction medium.

[0064] In some embodiments, the coverage of the microstructured surface of oxidized dextran can preferably be 1% - 25%. In some embodiments, collagen / gelatin and oxidized dex The concentration of dextran can preferably be about 1% to about 25%, more preferably 5 to 15%, and even more preferably 8 to 12%.

[0065] In some embodiments, the chemical coagulant can include one or more other substances that are deployed at a site where platelets are concentrated within the microstructure of the microstructured hemostatic agent. For example, they can include one or more substances that were present with the platelets. In some embodiments, the chemical additive can also include one or more salts such as phosphates, sodium salts, potassium salts, calcium salts, magnesium salts, and any other salts, or any combination of two or more of these.

[0066] Other exemplary substances, including but not limited to, saccharides such as monosaccharides and disaccharides (e.g., maltose, dextrose, mannose, trehalose, sucrose, polymers of sucrose, glucose); polysaccharides such as Ficoll-70 and Ficoll-400; glycerol; triglycerides; polysaccharides; lipids; dextran; polyvinylpyrrolidone (PVP); starch; hydroxyethyl starch (HES); etc. may be present in various embodiments of the microstructured surface.

[0067] Some embodiments can include biomolecules derived from human or animal sources, such as polypeptides (e.g., albumin such as bovine serum albumin and human serum albumin), casein, laminin, fibrinogen, etc.

[0068] Some embodiments of the microstructured hemostatic agent of the present disclosure can include hierarchically arranged microstructures. With this configuration, in addition to platelets, platelet microparticles can be concentrated. In such embodiments, the concentrated platelets can include about 10% to about 60% of the total number of particles, particularly platelets or platelet-derived particles, in the composition. For example, the platelets can include about 10% to about 50% of the particles, about 20% to about 50% of the particles, or about 30% to about 40% of the particles.

[0069] In other embodiments, about 70% of the platelet particles in the blood can be concentrated on the hierarchical surface, where the blood retains particles of typical platelet size. Thus, in some embodiments, up to about 70% of the particles are concentrated platelets present on a particular plane of the microstructured hemostat. Thus, the concentrated region of the microstructured hemostat can include 70% platelets and 30% microparticles, 60% platelets and 40% microparticles, 50% platelets and 50% microparticles, 40% platelets and 60% microparticles, 20% platelets and 80% microparticles, or 10% platelets and 90% microparticles.

[0070] Of course, any specific whole number of platelets or microparticles within any of the above ranges or amounts is contemplated by the present disclosure. One of ordinary skill in the art will likely immediately recognize a number of possible combinations of amounts of platelets and microparticles, and thus it is not necessary to specifically disclose each herein.

[0071] The platelet-concentrated region of the microstructured hemostat can be coated with a salt buffer that can include at least one saccharide to obtain a buffered platelet-containing region. The salt buffer can be any buffer that maintains at least a majority of the platelets in an intact functional state while in the buffer. The buffer can maintain the platelets at a pH of about 6 - 8, more preferably about 6.2 - about 7.8. The salt buffer can be an isotonic salt buffer that includes salts that platelets naturally encounter, such as sodium salts, potassium salts, calcium salts, etc., and combinations of such salts.

[0072] In some embodiments, the microstructured surface may include one or more salts that platelets do not naturally contact. The identity of the salt(s) in the buffer should not be present in an amount that is toxic to the platelets and cannot maintain at least a majority of the platelets in an intact functional state while in the buffer. Similarly, the buffering component can be any buffer that is non-toxic to the platelets and provides an appropriate buffering capacity to the composition at the temperature at which the composition is exposed during the methods of the present disclosure.

[0073] ​ Examples of buffers include any of the known commercially available biocompatible buffers such as Tris-based buffers such as HEPES, phosphate buffered saline (PBS), and TBS. Similarly, it may contain one or more of the following buffers: propane-1,2,3-tricarboxylic acid (tricarballylic acid); benzene pentacarboxylic acid; maleic acid; 2,2-dimethyl succinic acid; EDTA; 3,3-dimethyl glutaric acid; bis(2-hydroxyethyl)imino-tris(hydroxymethyl)-methane (BIS-TRIS); benzene hexacarboxylic acid (mellitic acid); N-(2-acetamido)imino-diacetic acid (ADA); butane-1,2,3,4-tetracarboxylic acid; pyrophosphoric acid; 1,1-cyclopentane diacetic acid (3 ,3 tetramethylene glutaric acid); 1, 4 piperazine bis-(ethanesulfonic acid) (PIPES); N-(2-acetamido)-2-aminoethanesulfonic acid (ACES); 1,1-cyclohexane diacetic acid; 3,6-endomethylene-1,2,3,6-tetrahydrophthalic acid (EMTA; ENDCA); imidazole; 2-(aminoethyl)trimethylammonium chloride (CHOLAMINE); N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES); 2-methylpropane-1,2,3-tricarboxylic acid (beta-methyltricarballylic acid); 2-(N-morpholino)propanesulfonic acid (MOPS); phosphoric acid; N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES); N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Further, the buffer system can provide buffering capacity in the range of pH 4 to pH 8.

[0074] The following examples are illustrative but not limiting.

Example

[0075] Pattern A Referring to FIG. 3, a microstructured hemostatic surface 300 is shown, which includes a sinusoidal surface 302 and complex pillars 304. In some embodiments, the sinusoidal surface can be arranged to include a triangular lattice. In some embodiments, the sinusoidal peaks can have a pitch of 1000 microns measured from one peak to an adjacent peak, and the sinusoidal peaks can include a peak height of 400 microns. The complex pillars 304 may be composed of a base pillar 306 and smaller pillars 308, and the smaller pillars may include longitudinal grooves 310 (see FIG. 3a). The base pillar 306 can be 25 microns in diameter and 45 microns in height. Further, the base pillar 306 can have a pitch of 75 microns measured from the center of one pillar to the center of an adjacent pillar and can be further arranged in a triangular lattice. The smaller pillars 308 can be arranged in a triangular lattice on the upper surface of the base pillar 306, and the smaller pillars 308 can be 5 microns in diameter and 15 microns in height with a pitch of 15 microns. The longitudinal grooves 310 can have a triangular cross-section and can include a width and / or height of 5 microns.

Example

[0076] Pattern B Referring to FIG. 4, the microstructured hemostatic surface 400 can include sinusoidal microstructures 402 that can include a height of 400 microns on a triangular lattice with a pitch of 1000 microns. Longitudinal parallel grooves 404 and transverse parallel grooves 406 can be placed on the hemostatic surface 400, and the grooves 404, 406 may be arranged orthogonally. In some embodiments, the grooves 404, 406 can have a width of 25 microns with a pitch of 50 microns from center to center. Longitudinal grooves 404 can, in some embodiments, include a depth of 50 microns. The lateral groove 406 can, in some embodiments, include a depth of 100 microns. In some embodiments, the grooves 404, 406 may be configured such that the depth arrangement of each groove creates pillars 408. In some embodiments, the pillars 408 may be squares with a length and width of 25 microns. In some embodiments, the upper surface of the pillar 408 can be machined to provide smaller pillars 412 disposed thereon. In some embodiments, the smaller pillars 412 can include pitches of 5 microns and 10 microns. In some embodiments, the smaller pillars 412 may be arranged in a square lattice, a triangular lattice, or randomly.

Example

[0077] Demonstration of Hemostasis Facilitated by Microstructure Purpose: Activated partial thromboplastin time (aPTT) is a test performed to investigate bleeding disorders and to monitor patients taking anticoagulants such as heparin that can inhibit factor X and thrombin while activating antithrombin. aPTT can be measured as a function of the textured surface structure of the embodiments disclosed in Examples 1 and 2 above.

[0078] Heparin was added (0.018 IU / 100 cc blood) to determine whether the microstructured hemostatic agent can locally reverse the effect of heparin.

[0079] Method: Fresh bovine blood was obtained and decalcified, and coagulation was prevented before the start of the test. The blood was collected in vials and decalcified by adding 0.01 M citrate. Plasma was separated and placed by centrifugation. During the test, the plasma was mixed with calcium ions to initiate the intrinsic pathway of the coagulation cascade. Kaolin (aluminum silicate hydrate) was added to activate the cascade. Kaolin activates contact-dependent factor XII. The partial thromboplastin time is the time it takes for a blood clot to form and is measured in seconds. Normally, a sample coagulates in 35 seconds on a smooth glass surface.

[0080] Test substance: (0.1 cc activated blood)

[0081] Embodiment of Example 1 - Polypropylene

[0082] Embodiment of Example 2 - Polypropylene

[0083] Results: When comparing the glass and the microstructured surface, the process of blood clot formation appears to proceed with the formation of small islands of blood clots, which then join together to form a macroscopic blood clot.

[0084] Based on these results, larger blood clots without heparin were studied in a static environment (rather than being stirred). On glass, normal blood coagulation appears to proceed through a series of solid-phase formations occurring over several size scales. This is evidence of fractal-dimensional behavior that can generally start at the 1-micron level. These islands join to form islands 10 - 25 microns in size, which then fuse into one at the 1000-micron scale.

[0085] In the case of heparin-added blood, these islands are not formed; rather, a gel is formed throughout the test volume without structure formation.

[0086] A microstructured surface with at least some structures having dimensions of 1000 microns can assist in the organization of blood clots over the entire range of the coagulation scale. [Table 1]

[0087] The clotting of normal blood on glass can proceed according to the fractal dimension scaling law. The clotting of heparinized blood does not seem to scale in the same dimension, and the entire blood volume can gel in the same time. As a result, an amorphous blood clot mass is formed instead of a fibrous mass.

[0088] Stirred heparinized blood produced an improved thromboplastin time on the microstructured surface compared to glass. When clotting is induced by a hierarchical microstructure, the clotting morphology can return to the normal fractal dimension scaling law for heparinized blood.

Example

[0089] Pattern C Referring to FIG. 5, the complex pillar surface 500 is composed of complex pillars 502. The complex pillars 502 are composed of base pillars 504 and smaller pillars 506. The base pillars 504 are arranged on a triangular lattice and can include a pitch of 100 microns. The base pillars 504 can also have a diameter of 40 microns. The smaller pillars 506 can be hierarchically arranged on the upper surface 508 of the base pillars 504. The smaller pillars 506 are also arranged in a triangular lattice and can have a pitch of 10 microns. The smaller pillars 506 can have a diameter of 5 microns.

Example

[0090] Demonstration of Hemostasis Promoted by Microstructure Purpose: The activated partial thromboplastin time (aPTT) is a test performed to investigate bleeding disorders and to monitor patients taking anticoagulants such as heparin that can inhibit factor X and thrombin while activating antithrombin. The aPTT is measured as a function of the textured surface and Gardenia fruit extract (coagulant).

[0091] Method: Fresh bovine blood was obtained and decalcified, and coagulation was prevented before starting the test. The blood was collected in vials and decalcified by adding 0.01 M citrate. Plasma was separated and placed by centrifugation. During the test, the plasma was mixed with calcium ions to initiate the intrinsic pathway of the coagulation cascade. Kaolin (aluminum silicate hydrate) was added to activate the cascade. Kaolin activates contact-dependent factor XII. The partial thromboplastin time is the time it takes for a blood clot to form and is measured in seconds. Usually, the sample coagulates in 35 seconds on a smooth glass surface.

[0092] Test substance: (0.1 cc activated plasma)

[0093] Glass plates containing and not containing 1% w / w gardenia extract; Embodiment 1 using polypropylene containing and not containing 1% w / w gardenia extract; Embodiment 4 using polypropylene containing and not containing 1% w / w gardenia extract.

[0094] Results: [Table 2] Examples

[0095] Composite microstructured hemostatic agent In some embodiments, a composite microstructured hemostatic agent can be used to correct coagulation deficiencies by removing serum from the bleeding surface and concentrating platelets and red blood cells near the wound-device interface.

[0096] Referring to FIG. 6, a composite microstructured hemostat 600 is shown. The hemostat 600 may include a first component 602 that can be composed of electrospun fibers 604. The electrospun fibers may be uniformly distributed on a second surface 606 that can be a modified pattern C (Example 4) in some embodiments. The electrospun fibers can be composed of an axial cross-section 608 having a diameter of 5 microns and a beaded cross-section 610 having a diameter of 30 microns. The electrospun component 602 can include a porosity of 1 to 2 microns. Preferably, the electrospun fibers 602 can be composed of a hydrophilic polymer. In one embodiment, for example, the electrospun fibers 602 can include absorbent polyurethane.

[0097] In some embodiments, the microstructured hemostat 600 can be used such that a blood droplet 612 can be composed of a liquid serum 614 and a solid 616 and contacts the microstructured hemostat 600. The solid 616 can include red blood cells, platelets, and / or microparticles. The first component 602 can have capillary contact with large pillars 606. The liquid serum 614 can be drawn between the large pillars 606, and the solid 616 can accumulate on small pillars 618. This arrangement can represent a Wenzel-Cassie interface that immobilizes the solid 616 within the first component 602 and promotes thrombus formation. The liquid serum 614 may be separated from the first component 604 to the second component 606.

[0098] The fluid component can be continuously drained from thrombus formation due to distal evaporation over a fairly large surface distal to the wound site. Optionally, a capillary reservoir can be created distal to the wound site for isolation.

Example

[0099] Pattern D In some embodiments, a composite microstructured hemostat can be used to reduce wound bleeding by shortening the time it takes for coagulation to occur. In one embodiment, this effect can be caused by removing serum from the bleeding surface and concentrating platelets and red blood cells near the wound-device interface.

[0100] In some embodiments, the hemostatic device can include a surface on which a hierarchical microstructure is disposed. In one embodiment, the hierarchical microstructure can include a first pillar and a second pillar, and the second pillar is disposed around the first pillar. The first pillar has a circular cross-section with a height of 200 microns, a diameter of 100 microns, and a pitch of 200 microns obtain. The second pillar can have a circular cross-section with a height of 20 microns, a diameter of 10 microns, and a pitch of 20 microns. When this embodiment is applied to an incision or wound site where bleeding occurs, bleeding stops at a faster rate than a surface without a hierarchical microstructure.

Example

[0101] Pattern E In some embodiments, the microstructured hemostat can utilize an electrospun frame surrounding the microstructured surface. In one embodiment, the frame of the hemostat can include 5-micron PET fibers that can be 500 microns thick. The frame may be disposed on a PLA backing. The PLA backing can include a hierarchical microstructure having a first pillar and a second pillar, with the second pillar disposed around the first pillar. The first pillar can have a circular cross-section with a height of 200 microns, a diameter of 100 microns, and a pitch of 200 microns. The second pillar can have a circular cross-section with a height of 20 microns, a diameter of 10 microns, and a pitch of 20 microns.

[0102] In some embodiments, the electrospun frame and the microstructured device disclosed above can contact blood and cause coagulation within about 2 minutes. Further, the blood can continue to flow through the microstructured layer and then contact the electrospun frame, "sucking in" the hemostatic agent to the target interface and causing coagulation at a predetermined position. In some embodiments, Blood flow can be reversed and backflowed through the incision or wound.

[0103] Accordingly, while specific embodiments of the present disclosure of novel and useful microstructured hemostatic agents have been described, such references are not intended to be construed as limitations on the scope of the present disclosure except as set forth in the following claims.

Claims

1. A device for promoting blood coagulation, comprising at least a first surface texture and a second surface texture, the second surface texture including a hierarchically arranged microstructured surface hierarchically disposed on the first surface texture, the first surface texture being disposed in plurality and spaced apart from each other on the microstructured surface, the second surface texture being disposed in plurality and spaced apart from each other on each of the plurality of the first surface textures, the first and second surface textures being configured to generate a plurality of spatially distributed surface energy gradients via a Wenzel-Cassie wetting state, a first portion of the microstructured surface generating a Cassie wetting state and a second portion of the microstructured surface generating a Wenzel wetting state, the first portion of the microstructured surface having a larger contact angle than the second portion, the hierarchically arranged microstructured surface being configured to have a fractal dimension between 2.06 and 2.08, the microstructured surface being configured to promote thrombus formation when in contact with blood, the hierarchically arranged microstructured surface being configured to have a shortened activated partial thromboplastin time compared to a surface lacking a hierarchical microstructure arrangement, device.

2. The hierarchically arranged microstructured surface further includes a third surface texture, the third surface texture being hierarchically arranged on the second surface texture, a plurality of the third surface textures being arranged separately from each other on each of the plurality of the second surface textures, the first surface texture being arranged in a triangular lattice, having a height in a direction perpendicular to the bottom of the microstructured surface of at least 400 micrometers, the second surface texture having a cylindrical shape, a diameter of 25 micrometers, and a height of 45 micrometers in a direction perpendicular to the upper surface of the first surface texture, the third surface texture having a cylindrical shape, a diameter of 5 micrometers, and a height of 15 micrometers in a direction perpendicular to the upper surface of the second surface texture, the device according to claim 1.

3. The device according to claim 2, wherein the second surface texture and the third surface texture are each arranged in a triangular lattice.

4. The device according to claim 2, wherein the third surface texture further includes a vertical surface with longitudinal grooves, each longitudinal groove having a triangular cross-sectional shape and having a width of 5 micrometers at the widest part of the longitudinal groove.

5. A device for separating blood components, including a hierarchically arranged microstructured surface, the hierarchically arranged microstructured surface including a first distinct surface and a second distinct surface, the first and second distinct surfaces each having a center, the center of the first distinct surface being located at least 50 micrometers from the center of the second distinct surface, the hierarchically arranged microstructured surface being configured to have a fractal dimension between 2.06 and 2.08, the first distinct surface being configured to localize a first blood component containing solids, the second distinct surface being configured to localize a second blood component containing fluid, The hierarchically arranged microstructured surface is configured to have a shortened activated partial thromboplastin time compared to a surface lacking a hierarchical microstructure arrangement. Device. **Claim 6** The first distinct surface includes at least two hierarchically arranged microstructured textures, the first distinct surface has a first fractal dimension, the second distinct surface includes at least two hierarchically arranged microstructured textures, the second distinct surface has a second fractal dimension, and the second distinct surface is configured to remove the second blood component by capillary action. The device according to claim 5. **Claim 7** The hierarchically arranged microstructured surface has the activated partial thromboplastin time, and the first distinct surface and the second distinct surface are configured as a whole. The device according to claim 6. **Claim 8** A device for promoting blood coagulation, including a hierarchically arranged microstructured surface, the hierarchically arranged microstructured surface is configured to have a fractal dimension between 2.06 and 2.08, the microstructured surface includes a hierarchical microstructure and a chemical coagulant, and the chemical coagulant is cross-linked to the microstructure. The hierarchically arranged microstructured surface is configured to have a shortened activated partial thromboplastin time compared to a surface lacking a hierarchical microstructure arrangement. Device. **Claim 9** The device according to claim 8, wherein the chemical coagulant includes gardenia fruit extract. **Claim 10** The device according to claim 8, wherein the chemical coagulant includes free amino groups of collagen / gelatin, and the microstructured surface includes a polymer substrate containing at least one aldehyde group. **Claim 11** The device according to claim 8, wherein the chemical coagulant includes at least one oxidized polysaccharide.

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