Devices for preparing platelet rich fibrin matrix and for dressing wounds, and kits and methods thereof

Abstract

Devices, kits, and methods provide enhanced blood separation to yield a composition of platelet rich fibrin matrix (PRFM). The devices may be pre-filled with layered components that facilitate the preparation of activation (e.g., full or partial activation) of platelet rich plasma (PRP) to produce a PRFM. The devices, kits, and methods reduce the number of steps involved with preparation of PRFM, reducing the need for external processing and mixing steps and reagents while providing a reproduceable methodology. The present disclosure further features devices, kits, and methods for providing dressings and uses thereof for localized and contained application of a biomaterial to a subject (e.g., the skin and / or a wound) for medical and / or cosmetic purposes. The devices disclosed herein can be used to promote drying and coagulation of the biomaterial, thereby forming a biomaterial dressing that remains applied upon removal of the wound dressing.

Description

Attorney Docket No. 005042-00013DEVICES FOR PREPARING PLATELET RICH FIBRIN MATRIX AND FOR DRESSING WOUNDS, AND KITS AND METHODS THEREOFPriority

[0001] The present application claims priority to U.S. Provisional Patent App. Nos.63 / 754,984, filed February 6, 2025, 63 / 768,644, filed March 7, 2025, and 63 / 929,563, filed December 2, 2025, the entire disclosures of which is incorporated herein by reference.Background

[0002] Wound healing is a significant focus of the medical field, as treatments are being developed to address various types of wounds, such as surgical wounds, lacerations, abrasions, bums, and traumatic injuries. Various materials, chemical and biological, are used for this purpose and several systems have been developed to ensure effective delivery of wound healing agents to the different layers of the skin and tissue.

[0003] Platelet rich fibrin matrix (PRFM) has been used for cosmetic purposes and for the treatment of injuries and conditions, in particular those relating to the healing and regeneration of skin, bone, and other tissue. PRFM is a fibrin matrix that contains a concentrated mixture of platelets and growth factors that play a role in the wound healing process.

[0004] There exists a need for improved devices, systems, and methods for the preparation and collection of PRFM, and devices and methods for producing an effective biomaterial dressing which improve the distribution and conformity of biomaterial on the wound.Summary of the Disclosure

[0005] The present disclosure features devices, methods, and kits for preparing PRFM from a blood sample. The present disclosure also features devices, kits, and methods for applying dressings and uses thereof for localized and contained application of a biomaterial to a subject (e.g., the skin and / or a wound) for medical and / or cosmetic purposes. The wound dressing disclosed herein can be used to promote drying and / or coagulation of the biomaterial, thereby forming a biomaterial dressing. The wound dressing disclosed herein may be applied to the wound before applying the biomaterial to facilitate placement of the biomaterial. The application of the biomaterial may be performed with an attached injection port to allow attachment of a container, such as a syringe. The wound dressing may also allow for ventilation of the wound during and following an application of the biomaterial.Attorney Docket No. 005042-00013The wound dressing may further be separable to allow removal of components, such as the injection port to provide comfort to the subject as the biomaterial solidifies and / or the wound heals. The biomaterial may be capable of remaining applied to skin even upon removal of the wound dressing.

[0006] A first aspect of the present disclosure is directed to a method comprising: introducing a blood sample into a tube, wherein the blood sample, when in the tube, is separated from a coagulating agent with at least one gel layer; centrifuging the tube to pass red blood cells of the blood sample through the at least one gel layer and to form a mixture of the coagulating agent and plasma of the blood sample positioned above the at least one gel layer; resuspending platelets of the blood sample into the mixture to form a platelet rich fibrin matrix (PRFM) layer in the tube; removing at least a portion of the PRFM layer from the tube in a fluid state with a syringe; applying a wound dressing at least partially over a wound; and injecting the at least the portion of the PRFM layer through an opening of the wound dressing, wherein air is vented through pores of the wound dressing.

[0007] In some embodiments, the method may include one or more of the following features. The at least the portion of the PRFM layer may be removed from the tube having a viscosity less than about 100 cP. The method may further include mixing a stabilizing agent with the coagulating agent and the plasma in tube. The method may further include mixing the blood sample with an anticoagulant in the tube before centrifuging. The centrifuging may be the only centrifugation. The injecting may be through a one-way valve of the wound dressing. Air may be vented through a plurality of first pores of a first layer of the wound dressing and one or more second pores of a second layer of the wound dressing. The injecting may be through an injection port attached to a distal surface of the wound dressing. The method may further include removing the injection port from the distal surface of the wound dressing after injecting. The removing the injection port may include lifting a tab of the injection port.

[0008] A second aspect of the present disclosure is directed to a kit comprising: a pre-filled tube configured to prepare a platelet rich fibrin matrix (PRFM) layer, wherein the pre-filled tube comprises: a first drug layer comprising a coagulating agent; a second drug layer comprising a stabilizing agent; and a gel layer separating the first drug layer and the second drug layer; and a wound dressing for applying a biomaterial to a subject, the wound dressing comprising: a first layer comprising a first opening and a plurality of first pores having a first width; and a second layer comprising a second opening and one or more second pores havingAttorney Docket No. 005042-00013a second width, wherein the first layer is configured to be between the second layer and the subject when the wound dressing is applied to the subject, the first opening is at least partially aligned with the second opening to allow passage of the biomaterial through the first layer and the second layer, and the second width is larger than the first width.

[0009] A third aspect of the present disclosure is directed to a method comprising: introducing a blood sample into a tube, wherein the blood sample, when in the tube, is separated from a stabilizing agent and a coagulating agent with at least one gel layer; centrifuging the tube to pass red blood cells of the blood sample through the at least one gel layer and to form a mixture of the stabilizing agent, the coagulating agent, and plasma of the blood sample positioned above the at least one gel layer; and resuspending platelets of the blood sample into the mixture to form a platelet rich fibrin matrix (PRFM) layer in the tube.

[0010] In some embodiments, the method of the third aspect may include one or more of the following features. The method may include removing at least a portion of the PRFM layer from the tube in a fluid state with a syringe; and applying the at least the portion of the PRFM layer to a wound site. The PRFM layer may be removed from the tube having a viscosity less than about 100 cP. The method may include removing at least a portion of the PRFM layer from the tube in a gel state; and applying the at least the portion of the PRFM layer to a wound site. The PRMF layer may be removed from the tube having a viscosity greater than about 100 cP. The coagulating agent may activate the platelets to form the PRFM layer, and the stabilizing agent stabilizes the PRFM layer. The at least one gel layer may include a first gel layer separating the coagulating agent from the stabilizing agent and a second gel layer separating the coagulating agent and the stabilizing agent from the blood sample. The method may include mixing the blood sample with an anticoagulant in the tube before centrifuging. The anticoagulant may include anticoagulant citrate dextrose A (ACD-A), buffered citrate, sodium citrate, citrate phosphate dextrose (CPD), ethylenediaminetetraacetic acid (EDTA), heparin, oxolate, a derivative thereof, or any combination thereof. The anticoagulant may be ACD-A. The stabilizing agent may be ascorbic acid. The coagulating agent may be a calcium salt. The calcium salt may be calcium gluconate and / or calcium chloride. The at least one gel layer may be at least one thixotropic gel. The tube may be prefilled with the stabilizing agent, the coagulating agent, and the at least one gel layer. The tube may be prefilled with an anticoagulant agent. The tube may contain a vacuum and is closed by a sealing cap. The stabilizing agent may be prefilled in the tube at a concentration of from about 400 mg / mL to about 600 mg / mL, inclusive. The coagulating agent may be prefilled in the tube at aAttorney Docket No. 005042-00013concentration of from about 50 mg / mL to about 150 mg / mL, inclusive. The centrifuging may be at an acceleration of from about 1000 g to about 4500 g, inclusive. The centrifuging may be for a time of from about 3 minutes to about 12 minutes, inclusive. The centrifuging may be the only centrifugation.

[0011] A fourth aspect of the present disclosure is directed to a pre-filled tube configured to prepare a platelet rich fibrin matrix (PRFM) layer, wherein the pre-filled tube comprises: a first drug layer comprising a coagulating agent; a second drug layer comprising a stabilizing agent; and a gel layer separating the first drug layer and the second drug layer.

[0012] In some embodiments, the pre-filled tube of the fourth aspect may include one or more of the following features. The pre-filled tube may comprise: a third drug layer comprising an anticoagulant; and a second gel layer separating the third drug layer from the first drug layer and the second drug layer. The anticoagulant may comprise anticoagulant citrate dextrose A (ACD-A), buffered citrate, sodium citrate, citrate phosphate dextrose (CPD), ethylenediaminetetraacetic acid (EDTA), heparin, oxolate, a derivative thereof, or any combination thereof. The anticoagulant may be ACD-A. The coagulating agent may comprise a calcium salt. The calcium salt may be calcium gluconate and / or calcium chloride. The coagulating agent may have a concentration of from about 50 mg / mL to about 150 mg / mL, inclusive. The stabilizing agent may be ascorbic acid. The stabilizing agent may have a concentration of from about 400 mg / mL to about 600 mg / mL, inclusive. The first drug layer may be closer to a distal end of the pre-filled tube than the second drug layer. The second drug layer may be closer to a distal end of the pre-filled tube than the first drug layer. The pre-filled tube may be sealed at a proximal end with a sealing cap to create a vacuum inside of the pre-filled tube. The pre-filled tube may contain an air pressure from about negative 50 kPa to about negative 101 kPa, inclusive.

[0013] A fifth aspect of the present disclosure is directed to a pre-filled tube configured to prepare a platelet rich fibrin matrix (PRFM) layer, wherein the pre-filled tube contains five layers of liquid, gel, or solid material and no more than five layers of liquid, gel, or solid material, the five layers of liquid, gel, or solid material comprising: a first drug layer comprising a coagulating agent; a second drug layer comprising a stabilizing agent; a third drug layer comprising an anticoagulant; a first gel layer separating the first drug layer and the second drug layer; and a second gel layer separating the third drug layer from the first drugAttorney Docket No. 005042-00013layer and the second drug layer, wherein the pre-filled tube is sealed at a proximal end with a sealing cap to create a vacuum inside of the pre-filled tube.

[0014] In some embodiments, the pre-filled tube of the fifth aspect may include one or more of the following features. The first drug layer may be closer to a distal end of the pre-filled tube than the second drug layer. The second drug layer may be closer to a distal of the prefilled tube than the first drug layer. The anticoagulant may comprise anticoagulant citrate dextrose A (ACD-A), buffered citrate, sodium citrate, citrate phosphate dextrose (CPD), ethylenediaminetetraacetic acid (EDTA), heparin, oxolate, a derivative thereof, or any combination thereof. The anticoagulant may be ACD-A. The coagulating agent may comprise a calcium salt. The calcium salt may be calcium gluconate and / or calcium chloride. The calcium salt may have a concentration of from about 50 mg / mL to about 150 mg / mL, inclusive. The stabilizing agent may be ascorbic acid. The stabilizing agent may have a concentration of from about 400 mg / mL to about 600 mg / mL, inclusive.

[0015] A sixth aspect of the present disclosure is directed to a pre-filled tube configured to prepare a platelet rich fibrin matrix (PRFM) layer, wherein the pre-filled tube contains five layers of liquid, gel, or solid material and no more than five layers of liquid, gel, or solid material, the five layers of liquid, gel, or solid material comprising: a first drug layer comprising calcium gluconate and / or calcium chloride in a concentration of about 50 mg / mL to about 150 mg / mL; a second drug layer comprising ascorbic acid in a concentration of from about 400 mg / mL to about 600 mg / mL; a third drug layer comprising an anticoagulant; a first gel layer separating the first drug layer and the second drug layer; and a second gel layer separating the third drug layer from the first drug layer and the second drug layer, wherein the pre-filled tube is sealed at a proximal end with a sealing cap to create a vacuum inside of the pre-filled tube.

[0016] A seventh aspect of the present disclosure is directed to a pre-filled tube configured to prepare a platelet rich fibrin matrix (PRFM) layer, wherein the pre-filled tube comprises: a first drug layer comprising a coagulating agent; a second drug layer comprising a stabilizing agent; a third drug layer comprising an anticoagulant; a first gel layer; and a second gel layer, wherein one of the first drug layer and the second drug layer has a distal end that contacts the pre-filled tube and a proximal end that contacts the first gel layer, another of the first drug layer and the second drug layer has a distal end that contacts the first gel layer and aAttorney Docket No. 005042-00013proximal end that contacts the second gel layer, and the third drug layer has a distal end that contacts the second gel layer and a proximal end that contacts a space in the pre-filled tube.

[0017] In some embodiments, the pre-filled tube of the seventh aspect may be sealed at a proximal end with a sealing cap to create a vacuum inside of the pre-filled tube.

[0018] In some embodiments of the disclosure, the pre-filled tube is a container sized to accommodate a volume of a blood sample of about 29 mL, wherein: (i) the volume of a first drug layer is about 2.0 mL; (ii) the volume of a first gel layer is about 4.3 mL; (iii) the volume of a second drug layer is about 2.0 mL; (iv) the volume of a second gel layer is about 4.3 mL; and (v) the volume of a third drug layer is about 3.2 mL; and wherein: (vi) the first drug layer includes a coagulating agent comprising calcium gluconate and / or calcium chloride, and the second drug layer includes a stabilizer comprising ascorbic acid, wherein a concentration of the calcium gluconate and / or calcium chloride is about 100 mg / mL, and wherein a concentration of the ascorbic acid is about 500 mg / mL; (vii) the first gel and the second are each a thixotropic gel; and (viii) the third drug layer includes an anticoagulant comprising ACD-A.

[0019] In some embodiments, the container is sized to accommodate a volume of the blood sample of about 32 mL, wherein: (i) the volume of a first drug layer is about 2.2 mL; (ii) the volume of a first gel layer is about 4.3 mL; (iii) the volume of a second drug layer is about 2.2 mL; (iv) the volume of a second gel layer is about 4.3 mL; and (v) the volume of a third drug layer is about 3.6 mL; and wherein: (vi) the first drug layer includes a coagulating agent comprising calcium gluconate and / or calcium chloride, and the second drug layer includes a stabilizer comprising ascorbic acid, wherein a concentration of the calcium gluconate and / or calcium chloride is about 100 mg / mL, and wherein a concentration of the ascorbic acid is about 500 mg / mL; (vii) the first gel layer and the second gel layer are each a thixotropic gel; and (viii) the third drug layer includes an anticoagulant comprising ACD-A.

[0020] In some embodiments: (i) the volume of a first drug layer is from about 0.5 mL to about 4 mL; (ii) the volume of the first gel layer is from about 0.5 mL to about 6 mL; (iii) the volume of the second drug layer is from about 0.5 mL to about 4 mL; (iv) the volume of the second gel layer is from about 0.5 mL to about 6 mL; and (v) the volume of the third drug layer is from about 0.1 mL to about 6 mL; and wherein: (vi) the first drug layer includes a coagulating agent comprising calcium gluconate and / or calcium chloride, and the second drug layer includes a stabilizer comprising ascorbic acid, wherein a concentration of theAttorney Docket No. 005042-00013calcium gluconate and / or calcium chloride is about 100 mg / mL, and wherein a concentration of the ascorbic acid is about 500 mg / mL; (vii) the first gel layer and the second gel layer are each a thixotropic gel; and (viii) the third drug layer includes an anticoagulant comprising ACD-A.

[0021] The disclosure features a kit for preparing a PRFM fraction from blood, including: (a) the pre-filled tube or container; and, optionally, one or more of the following: (b) a syringe including a needle; (c) a PRFM dispenser; and (d) a wound dressing.

[0022] An eighth aspect of the present disclosure is directed to a wound dressing for applying a biomaterial to a subject, the wound dressing comprising: a first layer comprising a first opening and a plurality of first pores having a first width; and a second layer comprising a second opening and one or more second pores having a second width, wherein the first layer is configured to be between the second layer and the subject when the wound dressing is applied to the subject, the first opening is at least partially aligned with the second opening to allow passage of the biomaterial through the first layer and the second layer, and the second width is larger than the first width.

[0023] In some embodiments, the wound dressing of the eighth aspect may include one or more of the following features. At least a subset of the first pores may be at least partially aligned with the one or more second pores to allow for ventilation through the first layer and the second layer. A distal surface of the second layer may be at least partially uncovered to allow for ventilation through the second layer directly to the atmosphere. The one or more second pores may be uncovered distally and directly open to the atmosphere. The first width may be from about 0.2 microns to about 500 microns, inclusive. The second width may be from about 1 mm to about 10 mm, inclusive. The first layer may be a mesh. The mesh may be non-absorbent. The wound dressing may include a valve configured for one-way passage of the biomaterial through the first opening and / or the second opening. The valve may be attached to the second layer. The valve may be a flap integral to the second layer and formed by a slit. A stop may be configured to limit distal movement of the valve. The second layer may have a first perimeter larger than a second perimeter of the second layer. An adhesive may be on a proximal surface of the second layer. The adhesive may adhere the first layer to the second layer. The adhesive may be on a peripheral portion of the second layer to adhere the second layer to the subject. The adhesive may comprise silicon. The wound dressing may include a non-adhesive insert positioned between the adhesive and the first layer, wherein the non-adhesive insert is configured to facilitate release of the second layer from the first layer.Attorney Docket No. 005042-00013The wound dressing may comprise an injection port attached to a distal surface of the second layer, wherein the injection port is configured to introduce the biomaterial through the first opening and the second opening. The injection port may be attached to the distal surface of the second layer with at least one adhesive. The at least one adhesive may include a first adhesive and a second adhesive, where the first adhesive is at least partially around the second opening, and the first adhesive is spaced apart from the second adhesive. The injection port may have a tab, and the second adhesive may adhere the tab to the second layer. The tab may be configured to allow a user to peel the injection port from the distal surface of the second layer. The tab may include a projection having an inner surface spaced apart from the distal surface of the second layer to allow the user to lift the tab from the distal surface of the second layer. The inner surface of the projection may be disposed at an acute angle with respect to the distal surface of the first layer. The at least one adhesive may be configured to releasably attach the injection port to the distal surface of the second layer. The at least one adhesive may have a distal surface configured to attach to the injection port and a proximal surface configured to attach to the distal surface of the second layer, and the distal surface of the at least one adhesive may have a greater tackiness than the proximal surface of the at least one adhesive to allow for release of the at least one adhesive from the distal surface of the second layer with the injection port. A kit may include the wound dressing; and a container configured to form or inject the biomaterial. The biomaterial may be a platelet rich plasma (PRP) or a platelet rich fibrin matrix (PRFM). The container may be a syringe configured to inject the biomaterial. The container may be a centrifuge tube including at least one separator gel configured to form the biomaterial.

[0024] A ninth aspect of the present disclosure is directed to a wound dressing for applying a biomaterial to a subject, the wound dressing comprising: a first layer comprising a first opening and a plurality of first pores having a first width; a second layer comprising a second opening, one or more second pores having a second width, and a flap configured to pivot relative to the second opening; and an injection port releasably attached to a distal surface of the second layer with a first adhesive and a second adhesive, wherein the first adhesive and the second adhesive are spaced apart, and the injection port is configured to introduce the biomaterial through the first opening and the second opening, wherein the first layer is configured to be between the second layer and the subject when the wound dressing is applied to the subject, the first opening is at least partially aligned with the second opening, the flap provides one-way passage of the biomaterial through the second opening, the secondAttorney Docket No. 005042-00013width is larger than the first width, and the one or more second pores are uncovered distally and directly open to the atmosphere.

[0025] In some embodiments, the wound dressing of the ninth aspect may include one or more of the following features. Each of the first adhesive and the second adhesive may have a distal surface configured to attach to the injection port and a proximal surface configured to attach to the distal surface of the second layer, and the distal surface may have a greater tackiness than the proximal surface to allow for release from the distal surface of the second layer with the injection port. The first adhesive may be at least partially around the second opening. The injection port may have a tab, and the second adhesive may attach the tab to the second layer. The tab may be configured to allow a user to peel the injection port from the distal surface of the second layer. The tab may comprise a projection having an inner surface spaced apart from the distal surface of the second layer to allow a user to lift the tab from the distal surface of the second layer. A kit may include the wound dressing; and a container configured to form or inject the biomaterial. The biomaterial comprises PRP or PRFM. The container comprises a syringe configured to inject the biomaterial. The container may be a centrifuge tube including at least one separator gel configured to form the biomaterial.

[0026] A tenth aspect of the present disclosure is directed to a method comprising: applying a wound dressing at least partially over a wound; and injecting a biomaterial through a first opening of a first layer of the wound dressing and a second opening of a second layer of the wound dressing to the wound, wherein air is vented through a plurality of first pores of the first layer and one or more second pores of the second layer.

[0027] In some embodiments, the method of the tenth aspect may include one or more of the following features. The injecting may be through a one-way valve of the wound dressing. The injecting may be through an injection port attached to a distal surface of the second layer. The method may further include removing the injection port from the distal surface of the second layer after injecting the biomaterial. The removing the injection port may include lifting a tab of the injection port. The method may include removing the second layer from the first layer after injecting the biomaterial. The method may include coagulating the biomaterial. The biomaterial may be platelet rich plasma (PRP) or platelet rich fibrin matrix (PRFM). The air may be vented from the second pores directly to the atmosphere.

[0028] The methods of preparing the PRP or PRFM may be combined with the method of applying the PRP and PRFM tube. Furthermore, the various embodiments of the pre-filledAttorney Docket No. 005042-00013tube and the various embodiments of the wound dressing may be combined in kits, with other components as disclosed herein.Definitions

[0029] To facilitate an understanding of this disclosure, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the disclosure. Terms such as “a,” “an,” and “the” are not intended to refer to only a singular entity; but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the disclosure, but their usage does not limit the disclosure, except as outlined in the claims.

[0030] As used herein, the term “about” refers to a value that is within 10% above or below the value being described.

[0031] As used herein, any values provided in a range of values include both the upper and lower bounds, and any values contained within the upper and lower bounds.

[0032] “Platelet poor plasma” or “PPP” refers to a blood-derived plasma product from which blood cells (e.g., platelets and red blood cells) have been substantially removed. Greater than 95% RBCs may be removed from the PPP. The PPP may include less than about 0.5% residual hematocrit (e.g., less than about 0.4% residual hematocrit or less than about 0.3% residual hematocrit). The PPP may be separated from a blood sample through centrifugation with one or more separator gels that separates blood components based on density.

[0033] “Platelet rich plasma” or “PRP” refers to a blood-derived product when platelets are resuspended in PPP. Due to the presence of the platelets, PRP may be stimulated to release growth factors and other proteins that promote healing. The PRP may have a liquid state and contain little to no fibrin, providing a rapid, short-term release of growth factors and other proteins. The viscosity of the PRP may be from about 1 cP to about 1.3 cP. The PRP may include at least about 2.5 x 105platelets / pL (e.g., at least 3 x 105platelets / pL, at least 3.5 x 105platelets / pL, at least 4 x 105platelets / pL, at least 4.5 x 105platelets / pL, or at least 5.5 x 105platelets / pL).

[0034] “Platelet rich fibrin matrix” or “PRFM” refers to a blood-derived product when PRP is activated to initiate a coagulation cascade, converting fibrinogen to fibrin. The PRFM may be activated by the addition of a coagulating agent such as thrombin or a calcium salt (e.g., calcium gluconate and / or calcium chloride). The formation of fibrin may lead to theAttorney Docket No. 005042-00013development of a fibrin matrix, which suspends the platelets, released growth factors, and other proteins that promote healing, allowing for a slower, sustained release of these bioactive molecules over the span of several days (e.g., from 1 day to a week, or time points therebetween). The fibrin matrix may form a gel-like matrix. As such, the PRFM may be used for localized treatments, particularly those needing a composition having a structure (e.g., gel-like matrix). The PRFM may have various degrees of coagulation, including PRFM in a fluid state and / or in a gel state. The viscosity of the PRFM may be about 1 cP to about 1000 cP and may be dependent on the degree of fibrin polymerization and platelet concentration. The PRFM may not be fully coagulated to a gel state when removed from the tube.Accordingly, the PRFM may have a viscous, fluid state that is still capable of being administered or applied with a syringe. For example, the PRFM may be removed from the tube with a syringe in a liquid state having a viscosity less than 100 cP. In some embodiments, the PRFM may be at least partially coagulated in the tube to be more viscous when administered or applied. For example, the PRFM may at least partially be in a gel state when removed. The PRFM may be removed from the tube having a viscosity greater than 100 cP. Once fully formed to a gel -like state (e.g, at the wound site), the PRFM may have a viscosity of up to 1000 cP.

[0035] “Treating” or “treatment” refers to the medical management of a patient with the intent that an amelioration, repair, or prevention of an injury or disease, pathological condition, or disorder will result. This term includes active treatment, that is, treatment directed specifically toward improvement of the injury or disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the injury or disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the injury or disease, pathological condition, or disorder; preventive treatment, that is, treatment directed to prevention of the injury or disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the injury or disease, pathological condition, or disorder.Brief Description of the Drawings

[0036] The following detailed description of the embodiments of the disclosure may be better understood when read in conjunction with the appended drawings. It should be understood,Attorney Docket No. 005042-00013however, that the disclosure is not limited to the precise arrangement and instrumentalities of the embodiments shown in the drawings.

[0037] FIGS. 1 A-1D illustrate a method of using a first exemplary embodiment of a pre-filled tube, according to the present disclosure.

[0038] FIG. 2 illustrates the pre-filled tube of FIG. 1A.

[0039] FIGS. 3A-3D illustrate a method of using a second exemplary embodiment of a prefilled tube, according to the present disclosure.

[0040] FIG. 4 illustrates a distal view of a first embodiment of a wound dressing of the present disclosure.

[0041] FIG. 5 illustrates a side view of the wound dressing of FIG. 4.

[0042] FIG. 6 illustrates an exploded perspective view of the wound dressing of FIGS. 4 and 5.

[0043] FIG. 7 illustrates an exploded side view of the wound dressing of FIGS. 4-6.

[0044] FIG. 8 illustrates a first proximal view of the wound dressing of FIGS. 4-7.

[0045] FIG. 9 illustrates a second proximal view of the wound dressing of FIGS. 4-8 with a liner removed.

[0046] FIG. 10 illustrates an exploded view of an injection port of the wound dressing of FIGS. 4-9.

[0047] FIG. 11 illustrates a cross-sectional view of a first connector of the injection port of FIG. 10.

[0048] FIG. 12 illustrates a perspective view of the connector of FIG. 11.

[0049] FIG. 13 illustrates a distal view of the connector of FIGS. 10-12.

[0050] FIG. 14 illustrates a second side view of the wound dressing of FIGS. 4-13.

[0051] FIG. 15 is a flow chart of a method of using the wound dressing of FIGS. 4-14

[0052] FIG. 16 illustrates an exploded perspective view of a second embodiment of a wound dressing.

[0053] FIG. 17 illustrates an exploded perspective view of the wound dressing of FIG. 16Attorney Docket No. 005042-00013

[0054] FIG. 18 illustrates a proximal view of the wound dressing of FIGS. 16 and 17 with a releasable liner removed.

[0055] FIG. 19 illustrates a distal view of a third embodiment of a wound dressing of the present disclosure.

[0056] FIG. 20 illustrates an exploded side view of the wound dressing of FIG. 19.

[0057] FIG. 21 illustrates a first perspective view of a fourth embodiment of a wound dressing of the present disclosure

[0058] FIG. 22 illustrates a second perspective view of the wound dressing of FIG. 21.

[0059] FIG. 23 illustrates a perspective cutaway view of the wound dressing of FIGS. 21 and 22.

[0060] FIG. 24 illustrates a perspective view of a fifth embodiment of a wound dressing of the present disclosure.

[0061] FIG. 25 illustrates a perspective view of a sixth embodiment of a wound dressing of the present disclosure.

[0062] FIG. 26 illustrates a perspective view of the wound dressing of FIG. 25 with a first container and a second container attached.

[0063] FIG. 27 illustrates a perspective view of a seventh embodiment of a wound dressing according to the present disclosure.

[0064] FIG. 28 illustrates a disassembled view of the wound dressing of FIG. 27.

[0065] FIG. 29 illustrates a second perspective view of the wound dressing of FIGS. 27 and 28.

[0066] FIG. 30 illustrates a perspective view of an eighth embodiment of a wound dressing of the present disclosure.

[0067] FIG. 31 illustrates a schematic of the wound dressing of FIG. 30.Detailed Description of the Drawings

[0068] The disclosure provides autologous wound care for a natural, safe, and effective approach for treating chronic and complex wounds. The disclosure may use a patient’s own cells and / or tissue to treat wounds, reducing the risk of immune rejection or allergic reactions. The disclosure provides devices, methods, and kits for preparation of platelet rich fibrin matrixAttorney Docket No. 005042-00013(PRFM). Particularly, the disclosure provides a pre-filled tube containing one or more drug layers separated by one or more gel layers. For example, the present disclosure provides prefilled tube that include three or five layers of liquid, gel, or solid. The pre-filled tube may minimize handling at the point of care and produce a stabilized platelet gel having an optimal release profile of growth factors (GF). The pre-filled tube may be a centrifuge tube configured to be spun to separate components of the blood. Blood components may then be mixed with one or more of the drug layers to form the PRFM.

[0069] The one or more drug layers may include a coagulating agent configured to initiate a coagulation cascade of PRP without fully transitioning the resulting PRFM to a coagulated state. The coagulating agents (e.g., calcium gluconate and / or calcium chloride) may precondition and / or activate the blood sample, causing insoluble fibrinogen to convert to fibrin, thereby promoting the coagulation cascade. The PRFM fraction may remain in a fluid state that may be readily extracted from the tube to be used for therapeutic and / or cosmetic purposes, for example with a syringe. The one or more drug layers may include a stabilizing agent (e.g., ascorbic acid) to preserve the viscosity of the PRFM and to ease handling of the PRFM. The coagulating agent and the stabilizing agent may mix with platelet poor plasma (PPP) of a blood sample during centrifugation. The PRFM may then be produced by resuspending platelets from the blood sample into the PPP mixed with the coagulating agent and the stabilizing agent. The one or more drug layers may further include an anti-coagulant to regulate the natural clotting pathways of the blood before centrifugation and contact with the coagulating agent. The one or more gel layers may separate the one or more drug layers in the tube to avoid mixing before centrifugation.

[0070] The pre-filled tube only requires a single centrifuge spin with all of the mixing performed inside of the centrifuge tube. However, in some embodiments, additional centrifuge spins may be applied if desired. The pre-filled tube reduces the procedural time and the number of steps involved with preparation of PRFM, avoiding the need for external processing and mixing reagents while providing a reproduceable methodology. The pre-filled tube reduces the need for calculations and handling of reagents at the point of care and avoids potential human error. The pre-loaded drug layers may be optimized to promote healing in ulcers (e.g., diabetic foot ulcers, pressure ulcers, or venous leg ulcers), for orthopedic applications (e.g., soft tissue repair, such as tendon or ligament repair, or to improve bone healing), as a hemostatic agent (e.g., a fibrin sealant), and / or for aesthetic and / or dermatologic applications (e.g., facial rejuvenation, hair restoration, scar healing orAttorney Docket No. 005042-00013improvement, and / or treatment of damaged skin and tissue, such as due to bums). Heat (e.g., body heat) may be used to fully activate the coagulation cascade of the resulting PRFM fraction, thereby forming, e.g., a PRFM gel. Additionally or alternatively, an agent or drug could be added to the separated PRFM fraction after centrifugation in order to initiate or expedite the coagulation cascade (e.g., to expedite formation of a PRFM gel).

[0071] In some embodiments, the coagulating agent includes a calcium salt, an aluminosilicate, a thrombin, a polyethylene glycol, a derivative thereof, or any combination thereof. In some embodiments, the calcium salt includes calcium gluconate, calcium chloride, calcium saccharate, a derivative thereof, or any combination thereof. In some embodiments, the calcium salt is calcium gluconate and / or calcium chloride. In some embodiments, the first drug layer includes from about 50 mg / mL to about 150 mg / mL (e.g., about 50 mg / mL, about 75 mg / mL, about 100 mg / mL, about 125 mg / mL, or about 150 mg / mL) of calcium gluconate and / or calcium chloride. In some embodiments, the stabilizing agent includes an ascorbic acid, sodium ascorbate, lactate, gluconic acid, and / or citrate. In some embodiments, the second drug layer includes from about 400 mg / mL to about 600 mg / mL (e.g., about 400 mg / mL, about 450 mg / mL, about 500 mg / mL, about 550 mg / mL or about 600 mg / mL) of ascorbic acid.

[0072] In some embodiments, the blood sample is centrifuged to separate an RBC fraction and to mix the coagulating agent and the stabilizing agent in the plasma of the blood sample. Platelets of the blood sample may then resuspended into the plasma mixture to form the PRFM. In some embodiments, the first gel layer and the second gel layer intermix to form a single gel layer that separates the plasma from the RBC fraction. In some embodiments, an amount of the coagulating agent, an amount of the stabilizing agent, and an amount of the anticoagulant controls an extent of activation of the platelets to form the PRFM fraction.

[0073] The present disclosure also features a device for dressing wounds. The device includes a wound dressing that may be useful for treating a wound (e.g., a burn wound, a traumatic wound, and / or a surgical wound) or an ulcer (e.g., a diabetic foot ulcer) by applying at least one of the devices described herein, e.g., using the method and kits described herein. The devices, methods, and kits can be used to deliver a biomaterial onto the skin and / or treatment site (e.g., wound or ulcer). The biomaterial may include PRP, PRFM, a coagulating agent, an antibiotic, an anesthetic, hyaluronic acid, a hydrogel, and / or an irrigation solution. The wound dressing can be used to contain the biomaterial and prevent exposure from the outside environment. Further, the wound dressing may provide ventilation of the biomaterialAttorney Docket No. 005042-00013to the outside, atmospheric environment. The wound dressing can be used to promote the gelation (or coagulation) of the biomaterial (e.g., PRP and / or PRFM) and / or serve as a mold. As such, the devices and methods herein described can be used to provide uniform delivery of the biomaterial over the treatment site to produce a conformable gel and / or to enhance the distribution and delivery of biologies (e.g., antibiotics, therapeutics, or other drugs) within the biomaterial to the treatment site in a controlled manner.

[0074] The various embodiments of the pre-filled tubes 100, 100’ and the various embodiments of the wound dressings 1000, 1000’, 2000, 3000, 4000, 4000’, 6000, 7000, 8000 may be used together in the methods and packaged together in kits in any combination, as discussed herein.

[0075] FIGS. 1 A-1D illustrate a method of using a first embodiment of a pre-filled container or tube 100, according to the present disclosure. As illustrated in FIG. 1 A, the pre-filled tube 100 may comprise a tube 102 with a proximal end 104 and a distal end 106. A sealing cap 130 may be disposed at the proximal end 104 of the tube 102. The tube 102 may contain a plurality of layers of material, such as five layers of material. From the distal end 106 to the proximal end 104, the tube 102 may contain a first drug layer 110, a first gel layer 120, a second drug layer 112, a second gel layer 122, and a third drug layer 114.

[0076] At least one of the first drug layer 110 and the second drug layer 112 may include a coagulating agent. At least one of the first drug layer 110, the second drug layer 112, and the third drug layer 114 may include a stabilizing agent. The third drug layer 114 may include an anticoagulant. For example, in some embodiments, the first drug layer 110 may comprise or consist of a coagulating agent, the second drug layer 112 may comprise or consist of a stabilizing agent, and the third drug layer may comprise or consist of an anticoagulant. In some embodiments, the first drug layer 110 may comprise or consist of the stabilizing agent, the second drug layer 112 may comprise or consistent of the coagulating agent, and the third drug layer 112 may comprise or consist of the anticoagulant. In some embodiments, the first drug layer 110 may comprise or consist of a first coagulating agent, the second drug layer 112 may comprise or consist of a second coagulating agent, and the third drug layer 114 may comprise or consist of the anticoagulant. The first coagulating agent and the second coagulating agent may be the same or different. The first gel layer 120 and the second gel layer 122 may comprise a thixotropic gel configured to separate the drug layers 110, 112, 114 before centrifugation. The gel layers 120, 122 may prevent any mixing between the coagulating agent, the stabilizing agent, and the anticoagulant prior to centrifugation. TheAttorney Docket No. 005042-00013remaining area within the tube 102 above the third drug layer 114 may be a space 108 to receive a blood sample 200.

[0077] The tube 102 may be prefilled as illustrated in FIG. 1 A and ready to be filled with the blood sample 200 by the user. FIG. 2 illustrates an isometric view of the pre-filled tubes 100. As illustrated in FIGS. 1A and 2, the tube 102 may be prefilled with the first drug layer 110, the first gel layer 120, the second drug layer 112, the second gel layer 122, and the third drug layer 114. Each layer 112-116, 120-122 may remain unperturbed despite the tube 102 being inverted, as illustrated. In some embodiments, the pre-filled tube 100 may be pre-filled with additional layers of liquid, gel, or solid, including bioactive drug layers and / or non-bioactive gel layers. In some embodiments, the pre-filled tube 100 may be pre-filled with only the five layers 110-114, 120-122 of liquid, gel, or solid (without any additional layers of material of liquid, gel, or solid (bioactive or non-bioactive) prior to filling with the blood sample 200).

[0078] After pre-filling, the sealing cap 130 (e.g., a stopper) may be inserted into the proximal end 104 of the tube 102 to provide an airtight (e.g., a vacuum) seal inside of the tube 102, as further illustrated. The sealing cap 130 may be configured to provide access to the space 108 inside of the tube 102 without disrupting the seal, for example by puncturing the sealing cap 130 with a needle of a syringe.

[0079] FIGS. 3A-3D illustrate a method of using a second embodiment of the pre-filled tube 100’ without the third drug layer 116 and the second gel layer 122. Thus, FIG. 3 A shows an embodiment of the pre-filled tube 100’, in which the tube 102 contains three layers of material. From the distal end 106 to the proximal end 104, the tube 102 may contain the first drug layer 110, the gel layer 120, and the second drug layer 112. The gel layer 120 may separate the first drug layer 110 and the second drug layer 112 before centrifugation. In some embodiments, the first drug layer 110 may comprise or consist of the coagulating agent, and the second drug layer 112 may comprise or consist of the anticoagulant. In some embodiments, the first drug layer 110 may comprise or consist of the coagulating agent, and the second drug layer 112 may comprise or consist of the stabilizing agent. The remaining volume within the tube 100 above the second drug layer 112 is space 108, which may be under vacuum when tube 102 is sealed. In some embodiments, the pre-filled tube 100 may be pre-loaded with additional layers of liquid, gel, or solid, including bioactive drug layers and / or non-bioactive gel layers. In some embodiments, the pre-filled tube 100 may be pre-loaded with only the three layers 110, 112, 120 of liquid, gel, or solid (without any additionalAttorney Docket No. 005042-00013layers of material of liquid, gel, or solid (bioactive or non-bioactive) prior to filling with the blood sample 200).

[0080] The entire disclosure of the first embodiment of FIGS. 1A-1D is expressly incorporated into the second embodiment of FIGS. 3A-3D for sake of brevity, except when otherwise indicated.

[0081] As illustrated in FIGS. IB, 3B, the space 108 in the tube 102 may be at least partially filled with the blood sample 200. The tube 102 may be filled with the blood sample 200 through a pressure differential from the vacuum of the space 108. For example, a needle assembly (e.g., a butterfly needle, not shown) may be used to transfer the blood sample 200 from a patient to the tube 102. The needle assembly may include a first needle that punctures skin of the patient and a second needle that punctures the sealing cap 130. A tubing may transfer the blood sample 200 from the first needle to the second needle. The second needle may be attached to a tube connector that receives the proximal end 104 of the tube 102 to align the second needle with the sealing cap 130. The size and / or the internal vacuum pressure of the tube 102 may be used to control the input of the blood sample 200, which may also be used to control the volume of PRFM.

[0082] As further illustrated, the blood sample 200 may mix with the material of an upper layer (not covered by a gel layer 120, 122). For example, the blood sample 200 may mix with the third drug layer 114 (as illustrated in FIG. IB) or the second drug layer 112 (as illustrated in FIG. 3B). The drug third drug layer 114 (FIG. 1 A) or the second drug layer 112 (FIG. 3 A) may include anticoagulant to prevent natural coagulation to occur when the blood sample 200 is first introduced into the tube 102. The anticoagulant may bind to calcium in the blood sample 200 to prevent the conversion of prothrombin to thrombin. After the blood sample 200 is introduced, the tube 102 may be inverted at least one time to mix the blood 200 with the anticoagulant prior to centrifugation. When initially filled, the gel layer(s) 120, 122 may separate the blood sample 200 from the first drug layer 110 and / or the second drug layer 112 to prevent mixing and / or activation through contact with the coagulating agent.

[0083] As illustrated in FIGS. 1C, 3C, the tube 102 may be centrifuged. The method may include a single centrifuging step. The centrifugation may mix and / or separate contents of the tube 102 into layers based on density. The gel layer(s) 120, 122 may liquefy and shift in response to the centrifugal force. The controlled movement of the gel layer(s) 120, 122 enables the mixing of the drug layers 110, 112, 114 with the blood sample 200. TheAttorney Docket No. 005042-00013configuration permits the mixing of the drug layers 110, 112, 114 with the plasma of the blood sample 200 through centrifugation, providing a convenient and reliable method for producing the PRFM without the risk of remixing with the other blood components. The mixing of the drug layers 112, 114 with the plasma may initiate a coagulation cascade. The coagulating agent may reverse or neutralize natural coagulants, inducing soluble proteins (e.g., fibrinogen) from the plasma to convert into fibrin to clot.

[0084] As illustrated, from the distal end 106 to the proximal end 104, the centrifugation may form a red blood cell (RBC) fraction layer 202, a gel layer 120 or 124, a platelet layer 204, and a plasma mixture (or PPP) layer 206 in the tube 102. Red blood cells of the blood 200 may have a higher density than the remaining components. Thus, during centrifugation, the red blood cells may separate from plasma of the blood 200, pass through the first gel layer 120 and / or the second gel layer 122, and may settle at the distal end 106 of the tube 102 in the RBC fraction layer 202. Platelets of the blood may have a density less than the first gel layer 120 and / or the second gel layer 122 but higher than plasma of the blood 200, the first drug layer 110, and / or the second drug layer 112. Thus, the platelets may separate from the plasma and not pass through the first gel layer 120 and / or the second gel layer 122. The plasma of the blood 200 may mix with the first drug layer 110 and / or the second drug layer 112 to form the plasma mixture layer 206 at the top of the layers. As illustrated in FIG. 1C, the first gel layer 120 and / or the second gel layer 122 may combine to form a gel layer 124 interface between the RBC fraction layer 202 and the platelet layer 204. For example, when the first gel layer 120 and the second gel layer 122 are the same material, the centrifugation may form a single, uniform gel layer 124.

[0085] As illustrated in FIGS. ID, 3D, the platelet layer 204 may be resuspended into the plasma mixture layer 206 to form a PRFM layer 208. For example, the cap 130 may be removed from the proximal end 104 of the tube 102, and a pipette of a syringe may be inserted into the tube. The syringe may be used to apply fluid pressure to the plasma mixture layer 206 to force the platelet layer 204 into the plasma mixture layer 206 and mix without disturbing the gel layer 120, 124 to keep the RBC layer 202 separated.

[0086] The resuspension of the platelets into the plasma mixture layer 206 may place the platelets in a state to further the coagulation cascade. Over time, the PRFM layer 208 may further coagulate and solidify to form a PRFM gel fraction 220. The platelets may be activated when mixed with the plasma mixture layer 206 to release coagulation factors andAttorney Docket No. 005042-00013proteins and to clot to form the PRFM gel fraction 220. The PRFM gel fraction 220 may start forming about 10 minutes after centrifuging and / or resuspending the platelets, depending on the type and / or concentrations of the drugs and / or volume of the blood sample 200.

[0087] Without being bound by any theory, the coagulating agent(s) may precondition the blood sample 200 and / or activate the platelets, which causes thrombin to initiate the release of fibrin, thereby promoting the coagulation cascade. The type of coagulating agent(s) may control the extent of activation of the coagulation cascade. The ratio of the amount of the one or more coagulating agents to the amount of the one or more anticoagulants, if present, may control the extent of activation of the PRFM layer 208 (and, accordingly, formation of PRFM). Furthermore, the ratio of the amount of the coagulating agent(s) and, optionally, anticoagulants to the volume of the blood sample 200 may also control the extent of activation of the PRFM layer 208. The amount of coagulating agent(s) may be controlled by the volume of the corresponding drug layer 110, 112 and the concentration of the drug(s) present in the drug layer.

[0088] The stabilizing agent may stabilize the PRFM and regulate the activation of the platelets. Without being bound by any theory, the stabilizing agent may maintain the structural integrity of the fibrin matrix. The stabilizing agent may provide tensile strength to the fibrin matrix. The stabilizing agent may further improve healing when applied to a wound by enhancing the stability of extracellular matrix (ECM), particularly for type I and III collagen, during the initial phases of healing.

[0089] The PRFM may be removed from the PRFM layer 208 with a biomaterial dispenser (e.g., a syringe and / or forceps) at various degrees of clotting, for example about 1 minute (or less) to about 20 minutes after centrifugation and / or resuspending the platelets. At least a portion of the PRFM layer 208 may be removed from the tube 102 when still in a viscous, liquid state. For example, at least a portion of the PRFM layer 208 may be removed from the tube 102 with a syringe shortly after resuspending the platelets and before substantially forming the PRFM gel fraction 220. For example, the PRFM layer 208 may be removed from the tube 102 within 10 minutes of centrifuging and / or resuspending the platelets. The PRFM layer 208 may then further coagulate at the wound site to form the PRFM gel fraction 220. The present disclosure allows the PRP or PRFM to be mixed quickly (e.g., by reducing transferring and / or mixing of reagents) to allow removal and application of the PRP or PRFM in a liquid state before substantially forming into the PRFM gel fraction 220, which can be performed at the wound site. Additionally or alternatively, the PRFM may be removed fromAttorney Docket No. 005042-00013the tube 102 after the PRFM layer 208 begins to solidify into the PRFM gel fraction 220, for example with forceps. For example, the PRFM layer 208 may be removed from the tube 102 after 10 minutes or later following the centrifuging and / or resuspending. After PRFM is removed, the PRFM may be applied to the wound, as discussed with reference to the method 500 and illustrated in FIG. 15.

[0090] The PRFM may facilitate healing of damaged tissue, promote regeneration of damaged tissue, and / or reduce scarring of the tissue (e.g., skin). For example, the PRFM may be applied to tissue for promoting healing in ulcers (e.g., diabetic foot ulcers, pressure ulcers, or venous leg ulcers), for orthopedic applications (e.g., soft tissue repair (e.g., tendon or ligament repair)), as a hemostatic agent, and / or aesthetic and / or dermatologic applications (e.g., facial rejuvenation, hair restoration, or scar improvement). The PRFM may be used topically (e.g., as a topical wound composition and / or as or with a dressing) or injected (e.g., injectable PRFM solution) into a site for treatment (e.g., a tissue site) or combined with the bone. The PRFM may be used as an autologous treatment with reduced preparation time and / or steps, for example with a single centrifugation and reduced transferring of blood components, for example by mixing the reagents and resuspending the platelets in the tube 102.

[0091] As further discussed below, the PRFM may also be used with a wound dressing (e.g., 1000) to apply over a wound site. In embodiments of wound treatment, the method may include applying the wound dressing over the wound to retain the PRFM at the wound site and / or promote healing. In some embodiments, the PRFM may be applied to the wound before the wound dressing is placed over the wound. In some embodiments, the wound dressing may be placed over the wound before the PRFM is applied to the wound. The wound dressing may include an opening in communication with a tubing, where a syringe filled with the PRFM may connect to a proximal end of the tubing. The syringe may then inject the PRFM through the opening of the wound dressing and onto the wound when the wound dressing is positioned over the wound.

[0092] The pre-filled tube 100, 100’ may be a component of a kit for preparing PRFM. The kit may be for cosmetic applications (e.g., hair restoration, facial rejuvenation, scar improvement, or anti-aging treatment), medical applications (e.g., wound (e.g., surgical, traumatic, or burn wounds) and / or tissue healing (e.g., soft tissue, skin, or bone healing) or,Attorney Docket No. 005042-00013e.g., treatment of an injury (e.g., musculoskeletal injuries) or a condition (e.g., osteoarthritis or tendonitis)).

[0093] The kit may include the needle assembly (not shown) to transfer the blood sample 200 from the patient to the tube 102. For example, the needle assembly (e.g., a butterfly needle) may include the first needle, the second needle, the tubing, and / or the tube holder. The first needle may be configured to puncture the skin of the patient to withdraw the blood sample 200. The second needle may be configured to puncture the sealing cap 130. The second needle may be attached to the tube connector that receives the proximal end 104 of the tube 102 to align the second needle with the sealing cap 130. The tubing may connect the first needle and the second needle to transfer the blood sample 200 from the patient to the prefilled tube 100, 100’.

[0094] The kit may include one or more syringes and / or needles to resuspend the platelets. The PRFM dispenser may include forceps, an additional tube, a vial, a reservoir, and / or any suitable fluid container configured to hold and dispense a biomaterial. The additional tube may include a syringe. The syringe may be connected to a needle. The needle may be used for insertion into sealing cap 130 to draw the PRP or PRFM from the tube 102. The additional tube may include a male Luer-lock plug disposed at one end of the tube configured to connect to the female Luer-lock threads of proximal region of a connector configured to engage with a proximal end 104 or sealing cap 130 of pre-filled tube 100.

[0095] In some embodiments, the PRFM dispenser includes an anticoagulant or a coagulating agent. In some embodiments, the kit further includes a vial including an anticoagulant. In some embodiments, the kit further includes a vial including a coagulating agent. In some embodiments, the kit further includes a tubing, a butterfly needle, a tube holder, and a second syringe, wherein the tube holder is connectable to the sealing cap of the container. In some embodiments, the kit further includes a PRFM storage tube. In some embodiments, the kit further includes a dish for containing a volume of the PRFM fraction for additional processing.

[0096] Additionally or alternatively, the kit may include the wound dressing, as discussed below (e.g., 1000). The wound dressing may be used with PRFM to treat a wound of a subject. The wound dressing may be applied to the subject (e.g., adhered or wrapped around) over the treatment site. The wound dressing may be conformable to the subject to allowAttorney Docket No. 005042-00013wearability. The wound dressing may be sterile. The wound dressing may adhere to the subject.Drug Layers 110, 112, 114

[0097] The disclosure includes at least one drug layer 110, 112, 114 in the tube 102. The first drug layer 110 and the second drug layer 112 may be the same or different (e.g., each drug layer may include a different drug, a different combination of drugs, or different amounts of the same drugs). The first drug layer 110 and the second drug layer 112 may have the same volume or different volumes. One of the drug layers 112, 114 may have a distal end of that contacts a gel layer 120, 122 and a proximal end that interfaces with the space 108. At least one of the drug layers 112 may have a proximal end and a distal end that each contact a gel layer 120, 122. One of the drug layers 110 may have a proximal end that contacts a gel layer 120, 122 and a distal end that contacts the tube 102. For example, in the embodiment of FIG.1A, the first drug layer 110 may have a distal end that contacts the distal end 106 of the tube 102. The second drug layer 112 may have a distal end that contacts the first gel layer 120 and a proximal end that contacts the second gel layer 122. The third drug layer 114 may have a distal end that contacts the second gel layer 122 and a proximal end that interfaces with the space 108. Similarly in the embodiment of FIG. 3 A, the first drug layer 110 may have a distal end that contacts the distal end 106 of the tube 102. The second drug layer 112 may have a distal end that contacts the gel layer 120 and a proximal end that interfaces with the space 108.

[0098] Upon introduction of the blood sample 200 to the pre-filled tube 100, the blood sample 200 may contact the proximal-most drug layer 112, 114 prior to centrifugation. At least one of the drug layers 110, 112 may be distal of the gel layer(s) 120, 122, such that, upon filling of the tube with a blood sample, the gel layer(s) 120, 122 separates the blood sample 200 from the drug layer(s) 110, 112. During centrifugation, the drug layer(s) 110, 112, 114 mix with plasma of the blood sample 200.

[0099] At least one of the first drug layer 110 and / or the second drug layer 112 may comprise the coagulating agent. Preferably, the coagulating agent is separated from the blood sample 200 by the gel layer(s) 120, 122 when initially introduced into the tube 102 (e.g., FIG. IB, 3B) to prevent premature coagulation. The coagulating agent(s) may be any agent capable of promoting coagulation of the blood sample 200 or its respective components during separation (e.g., the coagulating agent may be capable of promoting coagulation of the PPPAttorney Docket No. 005042-00013and / or PRP). Examples of a coagulating agent that may be included in a drug layer include a calcium salt, an aluminosilicate (e.g., kaolin), thrombin, polyethylene glycol, or a combination thereof. The calcium salt may be, e.g., calcium chloride, calcium gluconate, calcium saccharate, a derivative thereof, or combinations thereof. One or more of the drug layers may include a plurality of coagulating agents.

[0100] For example, the coagulating agent may be calcium gluconate and / or calcium chloride. The calcium gluconate and / or calcium chloride may be present in at least one of the drug layers 110, 112 at a concentration of from about 50 mg / mL to about 150 mg / mL (e.g., about 50 mg / mL, about 75 mg / mL, about 100 mg / mL, about 125 mg / mL or about 150 mg / mL). Calcium gluconate and / or calcium chloride may be included in at least one of the drug layers 110, 112 at a concentration that pre-activates the platelets without producing full coagulation (e.g., without causing the formation of a gel).

[0101] At least one of the first drug layer 110, the second drug layer 112, and the third drug layer 114 may comprise the stabilizing agent. The stabilizing agent may be ascorbic acid, sodium ascorbate, lactate, gluconic acid, and / or citrate. The stabilizing agent may be ascorbic acid present in at least one of the drug layers 110, 112, 114 at a concentration of from about lOOmg / mL to about 900 mg / mL, such as from about 400 mg / mL to about 600 mg / mL (e.g., about 400 mg / mL to about 500 mg / mL, about 500 mg / mL to about 600 mg / mL, about 450 mg / mL to about 550 mg / mL, e.g., about 400 mg / mL, about 450 mg / mL, about 500 mg / mL, about 550 mg / mL, or about 600 mg / mL, inclusive).

[0102] The first drug layer 110 and the second drug layer 112 may each have a volume of from about 0.5 mL to about 4 mL (e.g., from about 0.5 mL to about 1.0 mL, from about 0.5 mL to about 2.0 mL, from about 0.5 mL to about 3.0 mL, from about 0.5 mL to about 4.0 mL, from about 1.0 mL to about 2.0 mL, from about 1.0 mL to about 3.0 mL, from about 1.0 mL to about 4.0 mL, from about 2.0 mL to about 3.0 mL, from about 2.0 mL to about 4.0 mL, from about 3.0 mL to about 4.0 mL, e.g., about 0.5 mL, about 0.7 mL, about 1 mL, about 1.3 mL, about 1.5 mL, about 2.0 mL, about 2.2 mL, about 2.5 mL, about 3.0 mL, about 3.5 mL, or about 4.0 mL).

[0103] The first drug layer 110 may have a density of from about 1.0 g / mL to about 1.4g / mL (e.g., about 1.0 g / mL, about 1.1 g / mL, about 1.2 g / mL, about 1.3 g / mL, or about 1.4 g / mL). The second drug layer 112 may have a density of from about 1.0 g / mL to aboutAttorney Docket No. 005042-000131.4g / mL (e.g., about 1.0 g / mL, about 1.1 g / mL, about 1.2 g / mL, about 1.3 g / mL, or about 1.4 g / mL).

[0104] The ratio of the volume of the first drug layer 110 to the volume of the blood sample 200 may be from about 1 :4 to about 1 :30 (e.g., about 1 :4 to about 1:10, about 1 :4 to about 1:15, about 1 : 13 to about 1 : 16, about 1 : 10 to about 1 :20, about 1 :20 to about 1 :30, e.g., about 1:4. about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, or about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:21, about 1:22, about 1:23, about 1:24, or about 1:25, about 1:26, about 1:27, about 1:28, about 1:29, or about 1:30).

[0105] The ratio of the volume of the second drug layer 112 to the volume of the blood sample 200 may be from about 1 :4 to about 1 :30 (e.g., about 1 :4 to about 1:10, about 1 :4 to about 1:15, about 1:13 to about 1:16, about 1:10 to about 1:20, about 1:20 to about 1:30, e.g., about 1:4. about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:21, about 1:22, about 1:23, about 1:24, or about 1:25, about 1:26, about 1:27, about 1:28, about 1:29, or about 1:30).

[0106] The ratio of the combined volume of the first drug layer 110 and the second drug layer 112 to the volume of the blood sample 200 may be from 1 :2 to about 1:15 (e.g., about 1 :2 to about 1 : 10, about 1 :4 to about 1:15, about 1 : 12 to about 1:15, about 1 : 10 to about 1:15, about 1:6 to about 1:10, e.g., about 1:2, about 1:3, about 1:4. about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, or about 1:15).

[0107] When the blood sample 200 is centrifuged in the pre-filled tube 100, the ratio of the volume of the first drug layer 110 to the volume of the PRFM layer 208 may be from about 1 :2 to about 1:15 (e.g., about 1 :2 to about 1:5, about 1 :4 to about 1:10, about 1 :4 to about 1:15, about 1:7 to about 1:9, about 1:10 to about 1:15, e.g., about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, or about 1:15).

[0108] The ratio of the volume of the second drug layer 112 to the volume of the PRFM layer 208 may be from about 1 :2 to about 1:15 (e.g., about 1 :2 to about 1:5, about 1 :4 to about 1 : 10, about 1 :4 to about 1:15, about 1 :7 to about 1 :9, about 1 : 10 to about 1:15, e.g., about 1 :2,Attorney Docket No. 005042-00013about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, or about 1:15).

[0109] The ratio of the combined volume of the first drug layer 110 and the second drug layer 112 to the volume of the PRFM layer 208 may be from 1 : 1 to about 1:8 (e.g., about 1 : 1 to about 1 :3, about 1 :2 to about 1 :6, about 1 :4 to about 1:8, about 1 :2 to about 1 :4, about 1:5 to about 1:8, about 1:6 to about 1:8, e.g., about 1:1, about 1:2, about 1:3, about 1:4. about 1:5, about 1:6, about 1:7, or about 1:8).

[0110] The ratio of the volume of the first drug layer 110 to the volume of the second drug layer 112 may be about 1:1. The first drug layer 110 and second drug layer 112 may each have a volume of from about 1% to about 15% (e.g., about 1% to about 12%, about 1% to about 8%, about 1% to about 4%, about 2% to about 10%, about 2% to about 7%, about 4% to about 12%, about 4% to about 8%, about 6% to about 8%, about 6% to about 15%, about 10% to about 15%, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the volume of the PRP preparation tube.[oni] The third drug layer 114 (FIG. 1A) or the second drug layer 112 (FIG. 3 A) may comprise the anticoagulant (“anticoagulant layer”). The anticoagulant may provide more time for the user to use the PRFM prior to its full coagulation (e.g., formation of a solid gel).Alternatively, an anticoagulant layer may not be included, such as when the blood sample 200 drugs that inhibit the clotting or coagulation of PRP (e.g., blood thinners), as an anticoagulant may further elongate the process for pre-conditioning and / or activating the PRP. The type of anticoagulant(s) may control the extent of inhibition of the coagulation cascade. The ratio of the amount of the one or more coagulating agents to the amount of the one or more anticoagulants may also control the extent of activation of the PRP fraction. Furthermore, the ratio of the amount of each of the coagulating agent(s) and the anticoagulant(s) to the volume of the blood sample may also control the extent of activation of the PRP fraction. The amount of anticoagulant may be controlled by the volume of the anticoagulant layer and the concentration of the anticoagulant present in the anticoagulant layer.

[0112] The anticoagulant may be any agent capable of inhibiting, reducing, or downregulating coagulation of the blood or its respective components during separation (e.g., the anticoagulant may inhibit or reduce a coagulation cascade in the blood or in either of the PRP fraction and the RBC fraction). The distal end of the anticoagulant layer may contact a gelAttorney Docket No. 005042-00013layer, and the proximal end of the anticoagulant layer may interface the space 108 in the tube 102 such that, upon filling of the tube with the blood sample 200, the anticoagulant layer may contact the blood sample 200. For example, in some embodiments, the third drug layer 114 may comprise the anticoagulant (e.g., FIG. 1 A) and contact the second gel layer 122. In some embodiments, the second drug layer 112 may comprise the anticoagulant (e.g., FIG. 3 A) and contact the first gel layer 120. Upon introduction of the blood sample 200 into the tube 102, the proximal end of the anticoagulant layer may interface with the blood sample 200 prior to centrifugation. Prior to centrifugation, the tube may be inverted at least one time (e.g., at least 5 times, at least 6 times, or at least 7 times, e.g., 5 to 10 times, 7 to 10 times, e.g., 1 time, 2 time, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times) to mix the anticoagulant layer with the blood sample 200.

[0113] The anticoagulant may include buffered citrate, sodium citrate, citrate phosphate dextrose (CPD), ethylenediaminetetraacetic acid (EDTA), heparin, oxolate, anticoagulant citrate dextrose A (ACD-A), any derivative thereof, and combinations thereof. The anticoagulant may be ACD-A. ACD-A is composed of citric acid, sodium dextrose, and dextrose. ACD-A may include about 7 mg / mL citric acid, about 22 mg / mL sodium citrate, and about 24.5 mg / mL dextrose. The tube 102 may include a single anticoagulant layer that includes a plurality of anticoagulants. The anticoagulant layer may have a density of about 1.0 g / mL.

[0114] The anticoagulant layer may have a volume of from about 0.1 mL to about 6 mL (e.g., from about 0.1 mL to about 0.5 mL, from about 0.5 mL to about 1.0 mL, from about 0.5 mL to about 2.0 mL, from about 0.5 mL to about 3.0 mL, from about 0.5 mL to about 4.0 mL, from about 0.5 mL to about 5.0 mL, from about 0.5 mL to about 6.0 mL, from about 1.0 mL to about 2.0 mL, from about 1.0 mL to about 3.0 mL, from about 1.0 mL to about 4.0 mL, from about 1.0 mL to about 5.0 mL, from about 1.0 mL to about 6.0 mL, from about 1.5 mL to about 2.0 mL, from about 2.0 mL to about 3.0 mL, from about 2.0 mL to about 4.0 mL, from about 2.0 mL to about 5.0 mL, from about 2.0 mL to about 6.0 mL, from about 3.0 mL to about 4.0 mL, about 3.0 mL to about 5.0 mL, from about 3.0 mL to about 6.0 mL, from about 4.0 mL to about 4.5 mL, from about 4.0 mL to about 5.0 mL, from about 4.0 mL to about 6.0 mL, from about 5.0 mL to about 6.0 mL, e.g., about 0.1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.7 mL, about 1 mL, about 1.3 mL, about 1.5 mL, about 1.9 mL, about 2.0 mL, about 2.5 mL, about 3.0 mL, about 3.5 mL, about 4.0 mL, aboutAttorney Docket No. 005042-000134.25 mL, about 4.3 mL, about 4.5 mL, about 5.0 mL, about 5.5. mL, or about 6.0 mL). In some embodiments, the tube 102 does not include an anticoagulant layer.

[0115] The ratio of the volume of the anticoagulant layer to the volume of the blood sample 200 may be from about 1 :5 to about 1 : 100 (e.g., about 1 :5 to about 1:10, about 1 :5 to about 1:15, about 1 : 10 to about 1 :20, about 1 :5 to about 1 :50, about 1 : 50 to about 1 : 100, about 1 :25 to about 1:50, about 1:50 to about 1:75, e.g., about 1:100, about 1:95, about 1:90, about 1:85, about 1:80, about 1:75, about 1:70, about 1:65, about 1:60, about 1:55, about 1:50, about 1:45, about 1:40, about 1:35, about 1:30, about 1:25, about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, or about 1:5).

[0116] When centrifuged, the ratio of the volume of the anticoagulant layer to the volume of the PRFM layer 208 may be from about 1 :3 to about 1 :65 (e.g., about 1 :3 to about 1:5, about 1:3 to about 1:10, about 1:10 to about 1:20, about 1:5 to about 1:55, about 1:25 to about 1:50, about 1:30 to about 1:55, about 1:40 to about 1:65, e.g., about 1:65, about 1:60, 1:55, about 1:50, about 1:45, about 1:40, about 1:35, about 1:30, about 1:25, about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, or about 1:3).

[0117] The anticoagulant layer may have a volume of from about 1% to about 15% (e.g., about 1% to about 12%, about 1% to about 8%, about 1% to about 4%, about 2% to about 10%, about 2% to about 7%, about 4% to about 12%, about 4% to about 8%, about 6% to about 8%, about 6% to about 15%, about 10% to about 15%, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the volume of the tube 102.

[0118] The ratio of the volume of the anticoagulant to the volume of the other drug layers may be from about 1:3 to about 5:1 (e.g., about 1:3 to about 1:2, about 1:3 to about 1:1, about 1 :2 to about 1:1, about 1 : 1 to about 2:1, about 2: 1 to about 3:1, about 3 : 1 to about 4: 1, about 4:1 to about 5:1, e.g., about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1).

[0119] The ratio of the volume of the anticoagulant layer to the volume of the gel layer 120, 122 may be from about 1 :5 to about 3 : 1 (e.g., about 1 :5 to about 1 :3, about 1 :4 to about 1 :3, about 1 :4 to about 1 :2, about 1 :4 to about 1:1, about 1 :3 to about 1:1, about 1 :3 to about 1 :2, about 1 :2 to about 1:1, about 1 : 1 to about 2:1, about 2: 1 to about 3:1, e.g., about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, or about 3:1).Attorney Docket No. 005042-00013

[0120] When the pre-filled tube 102 includes five layers (see, e.g., FIG. 1A), the ratio of the volume of the third drug layer 114 to the volume of the first drug layer 110 or the second drug layer 112 may be from about 1:3 to about 5:1 (e.g., about 1:3 to about 1:2, about 1:3 to about 1:1, about 1 :2 to about 1:1, about 1 : 1 to about 2:1, about 2: 1 to about 3:1, about 3 : 1 to about 4:1, about 4:1 to about 5:1, e.g., about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4 : 1 , or about 5:1).

[0121] In some embodiments, the volume of the first drug layer 110 and the second drug layer 112 is the same. In some embodiments, the volume of the first drug layer 110 is different from the volume of the third drug layer 114. In some embodiments, the volume of the first drug layer 110 and the volume of the third drug layer 114 are each from about 0.5 mL to about 4.0 mL (e.g., from about 0.5 mL to about 1.0 mL, from about 0.5 mL to about 2.0 mL, from about 0.5 mL to about 3.0 mL, from about 0.5 mL to about 4.0 mL, from about 1.0 mL to about 2.0 mL, from about 1.0 mL to about 3.0 mL, from about 1.0 mL to about 4.0 mL, from about 2.0 mL to about 3.0 mL, from about 2.0 mL to about 4.0 mL, from about 3.0 mL to about 4.0 mL, e.g., about 0.5 mL, about 0.7 mL, about 1 mL, about 1.3 mL, about 1.5 mL, about 2.0 mL, about 2.2 mL, about 2.5 mL, about 3.0 mL, about 3.5 mL, or about 4.0 mL).

[0122] The pH of each of the one or more drug layer(s) 110, 112, 114 may be from about 5.5 to about 8.5 (e.g., about 5.6 to about 6.6, about 6 to about 8.2, about 6 to about 8.5, e.g., about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7., about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, or about 8.5).

[0123] The pH of the one or more drug layer(s) 110, 112 containing calcium gluconate and / or calcium chloride as the coagulating agent may be from about 6 to about 8.5 (e.g., about 6 to about 6.5, about 6.5 to about 7, about 7 to about 7.5, about 7.5 to about 8, about 8 to about 8.5, e.g., about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7., about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, or about 8.5).

[0124] The pH of the one or more drug layer(s) 110, 112, 114 containing ascorbic may be from about 5.6 to about 6.6 (e.g., about 5.6 to about 6.1, about 6.1 to about 6.6, e.g., about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, aboutAttorney Docket No. 005042-000136.4, about 6.5, or about 6.6). The pH of the one or more drug layer(s) containing an anticoagulant may be from about 4.5 to about 5.5 (e.g., about 4.5 to about 5, about 5 to about 5.5, e.g., about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, or about 5.5).

[0125] The weighted median pH is calculated by first determining the total volume of the solution (e.g. the total volume of all drug layers in the tube 102). For each component (e.g., layer) of the solution, the contribution of the overall pH is calculated by weighing its pH range endpoints (e.g., the minimum pH and the maximum pH) against the component’s (e.g., layer’s) volume fraction. Accordingly, the following equations may be used to calculate the weighted pH, where i represents the layer:

[0128] Each of the weighted pHs for the minimum pH is added together to provide the lower endpoint of the weighted pH range of the solution. Each of the weighted pHs for the maximum pH is added together to provide the upper endpoint of the weighted pH range of the solution. The weighted median pH is calculated in the same manner, using the median pH for each of the components (e.g., layers). Alternatively, the weighted median pH may be calculated by calculation of the median using the upper and lower endpoints of the weighted pH range.

[0129] The weighted pH of the combination of the one or more drug layer(s) 110, 112, 114 may be from about 4.5 to about 8 (e.g., about 4.5 to about 5, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7, about 7 to about 7.5, about 7.5 to about 8, about 6 to about 7.5, about 5 to about 6.5, about 5 to about 6, about 6 to about 7, about 4.5 to about 6, about 5 to about 7. e.g., about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8). The weighted pH of each of the one or more drug layer(s) may be from about 1.2 to about 4.5 (e.g., about 1.2 to about 1.5, about 1.5 to about 2, about 2 to about 2.5, about 2.5 to about 3, about 3 to about 3.5, about 3.5 to about 4, about 4 to about 4.5, e.g., about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7,Attorney Docket No. 005042-00013about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5).

[0130] The weighted pH of the anticoagulant layer may be from about 1.5 to 4 (e.g., about 1.5 to about 2, about 2 to about 2.5, about 2.5 to about 3, about 3 to about 3.5, about 3.5 to about 4, e.g., about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, or about 4).Gel Layers 120, 122

[0131] The gel layers 120, 122 may maintain separation between the one or more drug layers 110, 112, 114 prior to centrifugation of the tube 102. The gel layer 120, 122 may maintain separation of the blood sample 200 from the drug layers 112, 114 after filling the tube 102. The gel layer 120, 122 may further maintain separation of the RBC layer 202 after centrifugation.

[0132] The first gel layer 120 and / or the second gel layer 122 becomes less viscous when agitated or subjected to shear stress but returns to a more viscous state when at rest. During centrifugation, the gel layer 120, 122 allows the passage of RBCs through and beyond the gel, creating a distinct layer 120, 124 that separates the RBCs from the platelets and the plasma. The first gel layer 120 and / or the second gel layer 122 may be a thixotropic gel. In some embodiments, the first gel 120 and the second gel 122 are each a thixotropic gel.

[0133] The first gel layer 120 and the second gel layer 122 may be the same. Alternatively, the first gel layer 120 and the second gel layer 122 may be different (e.g., the gel layer may include two different gel components or the gel layers may have different thixotropic properties). The first gel layer 120 and the second gel layer 122 may intermix during centrifugation to form the gel layer 124 after centrifugation.

[0134] The first gel layer 120 and / or the second gel layer 122 may be a thixotrpic gel. The thixotropic gel may include petroleum hydrocarbon resin; tris(2-elthylhexyl) benzene- 1,2,4-tricarboxylate; silicon dioxide; silane, dichlorodimethyl-, reaction products with silica; polyolefin oligomer; polyacrylic acid; and / or polyamide.Attorney Docket No. 005042-00013

[0135] In some embodiments, the first gel layer 120 and / or the second gel layer 122 may have a density of from about 1.03 g / mLto about 1.7 g / mL (e.g., about 1.03 mg / mL, about 1.4 mg / mL, about 1.5 mg / mL, about 1.6 mg / mL, or about 1.7 mg / mL).

[0136] The first gel layer 120 and / or the second gel layer 122 may have a mass of from 0.5 g to about 12.0 g (e.g., from about 1 g to about 2 g, from about 2 g to about 3 g, from about 3 g to about 4 g, from about 3 g to about 5 g, from about 5 g to about 10 g, from about 8 g to about 10 g, from about 8 g to about 12 g, from about 3 g to about 9 g, e.g., about 1 g, about 1.5 g, about 2 g, about 2.5 g, about 3 g, about 3.5 g, about 4 g, about 4.5 g, about 5 g, about 5.5 g, about 6 g, about 6.5 g, about 7 g, about 7.5 g, about 8 g, about 8.5 g, about 9 g, about 9.5 g, about 10 g, about 10.5 g, about 11 g, about 11.5 g, or about 12 g).

[0137] The first gel layer 120 and / or the second gel layer 124 may a volume of from about 0.5 mL to about 6 mL (e.g., from about 0.5 mL to about 1.0 mL, from about 0.5 mL to about 2.0 mL, from about 0.5 mL to about 3.0 mL, from about 0.5 mL to about 4.0 mL, from about 0.5 mL to about 5.0 mL, from about 1.0 mL to about 2.0 mL, from about 1.0 mL to about 3.0 mL, from about 1.0 mL to about 4.0 mL, from about 1.0 mL to about 5.0 mL, from about 1.0 mL to about 6.0 mL, from about 2.0 mL to about 3.0 mL, from about 2.0 mL to about 4.0 mL, from about 2.0 mL to about 5.0 mL, from about 2.0 mL to about 6.0 mL, from about 3.0 mL to about 4.0 mL, about 3.0 mL to about 5.0 mL, from about 3.0 mL to about 6.0 mL, from about 4.0 mL to about 5.0 mL, from about 4.0 mL to about 6.0 mL, from about 5.0 mL to about 6.0 mL, e.g., about 0.5 mL, about 0.7 mL, about 1 mL, about 1.5 mL, about 2.0 mL , about 2.5 mL, about 3.0 mL, about 3.5 mL, about 4.0 mL, about 4.5 mL, about 5.0 mL, about 5.5. mL, or about 6.0 mL).

[0138] In some embodiments, the first gel layer 120 and / or the second gel layer 124 may have a combined volume of about 1 mL to about 12 mL (e.g., from about 1 mL to about 2 mL, from about 2 mL to about 3 mL, from about 3 mL to about 4 mL, from about 3 mL to about 5 mL, from about 5 mL to about 10 mL, from about 8 mL to about 10 mL, from about 8 mL to about 12 mL, from about 3 mL to about 9 mL, e.g., about 1 mL, about 1.5 mL, about 2 mL, about 2.5 mL, about 3 mL, about 3.5 mL, about 4 mL, about 4.5 mL, about 5 mL, about 5.5 mL, about 6 mL, about 6.5 mL, about 7 mL, about 7.5 mL, about 8 mL, about 8.5 mL, about 9 mL, about 9.5 mL, about 10 mL, about 10.5 mL, about 11 mL, about 11.5 mL, or about 12 mL). In some embodiments, the volume of the first gel layer 120 and the volume of the second gel layer 122 is the same, or the volume of the first gel layer is different than the volume of the second gel layer.Attorney Docket No. 005042-00013

[0139] The ratio of volume of the gel layer(s) 120, 122 to the volume of the blood sample 200 may be from about 1 :2 to about 1:10 (e.g., about 1 :2 to about 1 :4, about 1 :2 to about 1 :6, about 1 :2 to about 1 :7, about 1 :4 to about 1 :6, about 1 :5 to about 1 :7, about 1 :5 to about 1:10, about 1:7 to about 1:10, e.g., about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10).

[0140] The gel layer(s) 120, 122 may have a volume of from about 3% to about 25% (e.g., about 1% to about 12%, about 1% to about 8%, about 1% to about 4%, about 2% to about 10%, about 2% to about 7%, about 4% to about 12%, about 4% to about 8%, about 6% to about 8%, about 6% to about 15%, about 10% to about 15%, about 5% to about 20%, about 10% to about 20%, about 15% to about 20%, about 20 to about 25%, e.g., about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25%) of the volume of the tube 102.

[0141] In embodiments with a single gel layer 120 (see, e.g., FIG. 3A), the ratio of the volume of the first drug layer 110 or the second drug layer 112 to the gel layer 120 may be from about 1:7 to about 2:1 (e.g., about 1:7 to about 1:5, about 1:7 to about 1:6, about 1:7 to about 1:1, about 1:7 to about 1:4, about 1:5 to about 1:4, about 1:5 to about 1:3, about 1:5 to about 1 :2, about 1 :4 to about 1 :2, about 1 :4 to about 1 :3, about 1 :4 to about 1 : 1, about 1 :3 to about 1:2, about 1:3 to about 1:1, about 1:2 to about 1:1, about 1:1 to about 2:1, e.g., about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, or about 1:7).

[0142] In such embodiments with the single gel layer 120 (see, e.g., FIG. 3A), the first drug layer 110 may have a volume of about 0.5 mL to about 4 mL (e.g., from about 0.5 mL to about 1.0 mL, from about 0.5 mL to about 2.0 mL, from about 0.5 mL to about 3.0 mL, from about 0.5 mL to about 4.0 mL, from about 1.0 mL to about 2.0 mL, from about 1.0 mL to about 3.0 mL, from about 1.0 mL to about 4.0 mL, from about 2.0 mL to about 3.0 mL, from about 2.0 mL to about 4.0 mL, from about 3.0 mL to about 4.0 mL, e.g., about 0.5 mL, about 0.7 mL, about 1 mL, about 1.3 mL, about 1.5 mL, about 2.0 mL, about 2.2 mL, about 2.5 mL, about 3.0 mL, about 3.5 mL, or about 4.0 mL). The first drug layer 110 may have a density of about 1.0 g / mLto about 1.4 g / mL (e.g., about 1.0 g / mL, about 1.1 g / mL, about 1.2 g / mL, about 1.3 g / mL, or about 1.4 g / mL). When tube blood sample 200 is added to the pre-filled tube 100, the ratio of the volume of the first drug layer 110 to the volume of the blood sample 200 may be from about 1 :4 to about 1 :30 (e.g., about 1 :4 to about 1:10, about 1 :4 to about 1:15, about 1 : 13 to about 1 : 16, about 1 : 10 to about 1 :20, about 1 :20 to about 1 :30, e.g., aboutAttorney Docket No. 005042-000131:4. about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, or about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:21, about 1:22, about 1:23, about 1:24, or about 1:25, about 1:26, about 1:27, about 1:28, about 1:29, or about 1:30).Tube 102

[0143] The pre-filled tube 100, 100’ (referred collectively as 100, except when otherwise indicated) may be designed as a container defined by the tube 102.

[0144] The tube 102 may be any size or shape that is suitable for centrifugation (e.g., a centrifugation tube or a plasma preparation tube).

[0145] The tube 102 may have a volume of about 5 mL to about 60 mL (e.g., from about 5 mL to about 10 mL, from about 5 mL to about 15 mL, from about 15 mL to about 20 mL, from about 5 mL to about 40 mL, from about 9 mL to about 36 mL, from about 10 mL to about 40 mL, from about 10 mL to about 50 mL, from about 20 mL to 40 mL, from about 30 mL to about 35 mL, from about 30 mL to 40 mL, from about 20 mL to 30 mL, from about 50 mL to about 55 mL, or from about 50 mL to about 60 mL, e.g., about 5 mL, about 10 mL, about 15 mL, about 16 mL, about 20 mL, about 25 mL, about 30 mL, about 32 mL, about 35 mL, about 40 mL, about 45 mL, about 50 mL, about 54 mL, about 55 mL, or about 60 mL).

[0146] The tube 102 may be a 16 by 125 mm tube, a 20 x 125 mm tube, or a 29 x 115 mm tube.

[0147] The volume of the space (e.g., air) 108 within the tube 102 may be from about 4 mL to about 50 mL (e.g., about 5 mL to about 10 mL, about 10 mL to about 20 mL, about 20 mL to about 30 mL, about 30 mL to about 40 mL, about 40 mL to about 50 mL, about 10 mL to about 30 mL, about 15 mL to about 45 mL, e.g., about 5 mL, about 10 mL, about 11 mL, about 15 mL, about 20 mL, about 22 mL, about 25 mL, about 30 mL, about 34 mL, about 35 mL, about 40 mL, about 45 mL, or about 50 mL). The sealing cap may displace from about 0.5 mL to about 4 mL (e.g., from about 0.5 mL to about 1.0 mL, from about 0.5 mL to about 2.0 mL, from about 0.5 mL to about 3.0 mL, from about 0.5 mL to about 4.0 mL, from about 1.0 mL to about 2.0 mL, from about 1.0 mL to about 3.0 mL, from about 1.0 mL to about 4.0 mL, from about 2.0 mL to about 3.0 mL, from about 2.0 mL to about 4.0 mL, from about 3.0 mL to about 4.0 mL, e.g., about 0.5 mL, about 0.7 mL, about 1 mL, about 1.3 mL, about 1.5 mL, about 2.0 mL, about 2.2 mL, about 2.5 mL, about 3.0 mL, about 3.5 mL, or about 4.0 mL) of the volume of the tube 102.Attorney Docket No. 005042-00013

[0148] The tube 102 may be configured to draw from about 7 mL to about 22 mL of the blood sample 200 (e.g., from about 7 mL to about 10 mL, about 10 mL to about 18 mL, about 8 mL to about 12 mL, from about 18 mL to about 20 mL, from about 19 mL to about 20 mL, from about 20 mL to about 22 mL, e.g., about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, or about 22 mL of the blood sample 200) may have a pressure of between about negative 90 kPa and about negative 100 kPa (e.g., about negative 90 kPa, about negative 91 kPa, about negative 92 kPa, about negative 93 kPa, about negative 94 kPa, about negative 95 kPa, about negative 96 kPa, about negative 97 kPa, about negative 98 kPa, about negative 99 kPa, or about negative 100 kPa, e.g., about negative 90 to about negative 95 kPa or about negative 95 kPa to negative 100 kPa).

[0149] Negative pressure or vacuum in the tube 102 may be maintained by the sealing cap 130. The negative pressure of the tube 102 may be from about negative 50 kPa to about negative 101 kPa (e.g., about negative 80 kPa to about negative 100 kPa, e.g., about negative 50 kPa, about negative 55 kPa, about negative 60 kPa, about negative 65 kPa, about negative 70 kPa, about negative 75 kPa, about negative 80 kPa, about negative 85 kPa, about negative 86 kPa, about negative 87 kPa, about negative 88 kPa, about negative 89 kPa, about negative 90 kPa, about negative 91 kPa, about negative 92 kPa, about negative 93 kPa, about negative 94 kPa, about negative 95 kPa, about negative 96 kPa, about negative 97 kPa, about negative 98 kPa, about negative 99 kPa, about negative 100 kPa, or about negative 101 kPa).

[0150] The interior of the tube 102 may include a coating (e.g., a silicone coating).

[0151] The tube 102 may be configured to draw from about 10 mL to about 36 mL of the blood sample 200 (e.g., from about 10 mL to about 30 mL, from about 10 mL to about 20 mL, from about 15 mL to about 30 mL, from about 15 mL to about 20 mL, from about 30 mL to about 33 mL, from about 33 mL to about 36 mL, from about 32 mL to about 36 mL, from about 34 mL to about 36 mL, from about 30 mL to about 32 mL, or from about 35 mL to about 36 mL, e.g., about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, about 30 mL, about 31 mL, about 32 mL, about 33 mL, about 34 mL, about 35 mL, or about 36 mL of the blood sample 200) may have a negative pressure of between negative 80 kPa and negative 90 kPa (e.g., about negative 80 kPa, about negative 81 kPa, about negative 82Attorney Docket No. 005042-00013kPa, about negative 83 kPa, about negative 84 kPa, about negative 85 kPa, about negative 86 kPa, about negative 87 kPa, about negative 88 kPa, about negative 89 kPa, or about negative 90 kPa, e.g., about negative 80 to about negative 85 kPa or about negative 85 kPa to negative 90 kPa).

[0152] The tube 102 may be configured to draw from about 14 mL to about 40 mL of the blood sample 200 (e.g., from about 14 mL to about 20 mL, from about 15 mL to about 30 mL, from about 15 mL to about 25 mL, from about 25 mL to about 30 mL, from about 20 mL to about 30 mL, from about 31 mL to about 35 mL, from about 35 mL to about 38 mL, from about 38 mL to about 40 mL, from about 34 mL to about 35 mL, from about 34 mL to about 36 mL, from about 34 mL to about 38 mL, from about 36 mL to about 40 mL, e.g., about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, about 30 mL, about 31 mL, about 32 mL, about 33 mL, about 34 mL, about 35 mL, about 36 mL, about 37 mL, about 38 mL, about 39 mL, or about 40 mL of the blood sample 200) may have a negative pressure of between negative 90 kPa and negative 100 kPa (e.g., about negative 90 kPa, about negative 91 kPa, about negative 92 kPa, about negative 93 kPa, about negative 94 kPa, about negative 95 kPa, about negative 96 kPa, about negative 97 kPa, about negative 98 kPa, about negative 99 kPa, or about negative 100 kPa, e.g., about negative 90 to about negative 95 kPa or about negative 95 kPa to negative 100 kPa).

[0153] In some embodiments, the tube 102 further contains the blood sample 200. In some embodiments, the tube 102 includes a volume of from about 5 mL to about 60 mL, and a ratio of the volume of the first gel layer 120 to a volume of the blood sample 200 is from about 1 :4 to about 1 :20 (e.g., about 1 :4 to about 1:8, about 1 :4 to about 1 : 12, about 1 :4 to about 1:16, about 1 :6 to about 1:8, about 1 :6 to about 1:10, about 1 :6 to about 1:14, about 1 :6 to about 1 :20, about 1 : 10 to about 1 : 14, about 1 :8 to about 1:16, about 1 : 16 to about 1 :20, e.g., about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, or about 1:20).

[0154] In some embodiments, the tube 102 includes a volume of from about 5 mL to about 60 mL, and a ratio of the volume of the second gel layer 122 to the volume of the blood sample 200 is from about 1 :4 to about 1 :20 (e.g., about 1 :4 to about 1:8, about 1 :4 to about 1 : 12, about 1 :4 to about 1:16, about 1 :6 to about 1:8, about 1 :6 to about 1:10, about 1 :6 toAttorney Docket No. 005042-00013about 1 : 14, about 1 :6 to about 1 :20, about 1 : 10 to about 1 : 14, about 1 :8 to about 1:16, about 1:16 to about 1:20, e.g., about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, or about 1:20).

[0155] In some embodiments, the tube 102 includes a volume of from about 5 mL to about 60 mL, and a ratio of a combined volume of the first gel layer 120 and the second gel layer 122 and the volume of the blood sample 200 is from about 1 :2 to about 1:10 (e.g., about 1 :2 to about 1 :4, about 1 :2 to about 1 :6, about 1 :2 to about 1 :7, about 1 :4 to about 1 :6, about 1:5 to about 1:7, about 1:5 to about 1:10, about 1:7 to about 1:10, e.g., about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10).

[0156] In some embodiments, the tube 102 may include a volume of from about 5 mL to about 60 mL, and a ratio of the volume of the first drug layer 110 to the volume of the blood sample 200 is from about 1 :4 to about 1 :30 (e.g., about 1 :4 to about 1:10, about 1 :4 to about 1:15, about 1:13 to about 1:16, about 1:10 to about 1:20, about 1:20 to about 1:30, e.g., about 1:4. about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, or about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:21, about 1:22, about 1:23, about 1:24, or about 1:25, about 1:26, about 1:27, about 1:28, about 1:29, or about 1:30).

[0157] In some embodiments, the tube 102 may include a volume of from about 5 mL to about 60 mL, and a ratio of the volume of the second drug layer 112 to the volume of the blood sample 200 is from about 1 :4 to about 1 :30 (e.g., about 1 :4 to about 1:10, about 1 :4 to about 1:15, about 1:13 to about 1:16, about 1:10 to about 1:20, about 1:20 to about 1:30, e.g., about 1:4. about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, or about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:21, about 1:22, about 1:23, about 1:24, or about 1:25, about 1:26, about 1:27, about 1:28, about 1:29, or about 1:30).

[0158] In some embodiments, the tube 102 includes a volume of from about 5 mL to about 60 mL, and a ratio of a combined volume of the first drug layer 110 and the second drug layer 112 to the volume of the blood sample 200 is from about 1:2 to about 1:15 (e.g., about 1:2 to about 1 : 10, about 1 :4 to about 1:15, about 1 : 12 to about 1:15, about 1 : 10 to about 1:15, about 1:6 to about 1:10, e.g., about 1:2, about 1:3, about 1:4. about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, or about 1:15).Attorney Docket No. 005042-00013

[0159] In some embodiments, the tube 102 may include a volume of from about 5 mL to about 60 mL, and a ratio of the volume of the third drug layer 114 to the volume of the blood sample 200 is from about 1:5 to about 1:100 (e.g., about 1:5 to about 1:10, about 1:5 to about 1:15, about 1 : 10 to about 1 :20, about 1 :5 to about 1 :50, about 1 : 50 to about 1 : 100, about 1 :25 to about 1:50, about 1:50 to about 1:75, e.g., about 1:100, about 1:95, about 1:90, about 1:85, about 1:80, about 1:75, about 1:70, about 1:65, about 1:60, about 1:55, about 1:50, about 1:45, about 1:40, about 1:35, about 1:30, about 1:25, about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, or about 1:5).

[0160] In some embodiments, the tube 102 may include a volume of from about 5 mL to about 60 mL, and a ratio of a combined volume of the first drug layer 110, the second drug layer 112, and the third drug layer 114 to the volume of the blood sample 200 is from about 1 : 1 to about 1:10 (e.g., about 1 :2 to about 1:10, about 1 :4 to about 1:8, about 1 :7 to about 1:10, about 1:2 to about 1:8, about 1:1 to about 1:5, e.g., about 1:1, about 1:2, about 1:3, about 1:4. about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10).

[0161] The tube 102 may be a 16 x 125 mm tube, a 20 x 125 mm tube, or a 29 x 115 mm tube. The tube 102 may be any commercially available tube for the preparation of platelet rich plasma.

[0162] The sealing cap 130 may be at the proximal end of the tube 102. The sealing cap 130 may be configured for insertion into the proximal end of the tube in order to seal the tube. For example, the sealing cap may be configured to provide an airtight seal, such that the tube may be placed under vacuum pressure. The sealing cap 130 may also be made from a pierceable material, such as a thermoplastic elastomer (TPE), such as rubber (e.g., bromo butyl rubber), that may be easily pierced, e.g., with a needle and / or syringe.

[0163] The sealing cap 130 may be pierced multiple times throughout the preparation of PRFM without it being removed from the tube 102 (e.g., by syringe injection or other known methods). The needle and / or syringe may be configured to inject the blood sample 200 into the tube 102. For example, a syringe may be connected to another needle (e.g., a butterfly needle), e.g., by way of tubing, for receiving a blood sample as the blood is drawn from a subject. In this example, a tube holder may be equipped with the pre-filled tube 100 for compatibility with the additional components (e.g., needle, tubing, and / or syringe) for drawing blood, such that, as the blood sample 200, it is transferred directly into the tube 102.Attorney Docket No. 005042-00013The sealing cap 130 may also be pierced (e.g., by a needle) for removal of the PFRM after centrifugation.Process

[0164] The volume of the blood sample 200 added to the pre-filled tube 100 may be from about 2 mL to about 45 mL (e.g., about 2 mL to about 9 mL, about 5 mL to about 10 mL, about 5 mL to about 20 mL, about 5 mL to about 30 mL, about 5 mL to about 40 mL, about 10 mL to about 40 mL, about 20 mL to about 40 mL, about 30 mL to about 40 mL, about 10 mL to about 30 mL, about 30 mL to about 45 mL, about 20 mL to about 30 mL, about 15 mL to about 30 mL, about 10 mL to about 45 mL, e.g., about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, about 30 mL, about 31 mL, about 32 mL, about 33 mL, about 34 mL, about 35 mL, about 36 mL, about 37 mL, about 38 mL, about 39 mL, about 40 mL, about 41 mL, about 42 mL, about 43 mL, about 44 mL, or about 45 mL).

[0165] The centrifugation may be at an acceleration of about 1000 g to about 4500 g (e.g., about 1200 g to about 3500 g, about 1220 g to about 3000 g, about 1220 g to about 2500 g, about 1220 g to about 1500 g, about 1220 g to about 1250 g, about 1500 g to about 3500 g, about 1500 g to about 3000 g, about 1500 g to about 1750 g, about 1750 g to about 2500 g, about 1750 g to about 2250 g, about 1750 g to about 2000 g, about 2000 g to about 2500 g, about 2250 g to about 2500 g, e.g., about 1220 g, about 1200 g, about 1300 g, about 1400 g, about 1500 g, about 1600 g, about 1700 g, about 1800 g, about 1900 g, about 2000 g, about 2100 g, about 2200 g, about 2300 g, about 2400 g, or about 2500 g).

[0166] The centrifugation may be for a time between about 3 minutes to about 12 minutes (e.g., about 3 minutes to about 10 minutes, about 5 minutes to about 8 minutes, about 6 minutes to about 12 minutes, about 6 minutes to 10 minutes, about 8 minutes to 12 minutes, about 8 minutes to 10 minutes, about 10 minutes to about 12 minutes or, e.g., about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, or about 12 minutes).PRFM LAYER 208Attorney Docket No. 005042-00013

[0167] The PRFM layer 208 may be about 45% to about 65% (e.g., about 45%, about 50%, about 55%, about 60%, or about 65%) of the volume of the blood sample 200. The percentage of PRP layer 208 in the blood sample 200 of the subject may be subjectdependent.

[0168] The PRFM layer 208 prepared in the tube 102 may have a volume from about 1 mL to about 30 mL (e.g., about 1 mL to about 5 mL, about 5 mL to about 10 mL, about 10 mL to about 15 mL, about 15 mL to about 20 mL, about 20 mL to about 25 mL, about 25 mL to about 30 mL, about 5 mL to about 20 mL, about 10 mL to about 20 mL, about 15 mL to about 25 mL, e.g., about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, or about 25 mL).

[0169] In some embodiments, the volume of the PRFM layer 208 may be from 1 mL to about 8 mL (e.g., from about 1 mL to about 4 mL, from about 3 mL to about 7 mL, or from about 4 mL to about 6 mL, e.g., about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, or about 8 mL) when the tube 102 is configured to draw from about 2 mL to about 12 mL (e.g., about 2 mL to about 10 mL, about 2 mL to about 8 mL, about 2 mL to about 4 mL, about 4 mL to about 8 mL, about 4 mL to about 12 mL, about 8 mL to about 10 mL, about 10 mL to about 12 mL, e.g., about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, or about 12 mL of the blood sample 200).

[0170] In some embodiments, the volume of the PRFM layer 208 may be from about 3 mL to about 13 mL (e.g., from about 3 mL to about 8 mL, from about 8 mL to about 12 mL, or from about 9 mL to about 11 mL, e.g., about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, or about 13 mL) when pre-filled tube 100 100 is configured to draw from about 7 mL to about 22 mL (e.g., from about 7 mL to about 10 mL, about 10 mL to about 18 mL, about 8 mL to about 12 mL, from about 18 mL to about 20 mL, from about 19 mL to about 20 mL, from about 20 mL to about 22 mL, e.g., about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, or about 22 mL of the blood sample 200).Attorney Docket No. 005042-00013

[0171] In some embodiments, the volume of the PRFM layer 208 may be from about 6 mL to about 20 mL (e.g., from about 6 mL to about 13 mL, from about 13 mL to about 17 mL, from about 16 mL to about 20 mL, or from about 14 mL to about 16 mL, e.g., about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, or about 20 mL) when pre-filled tube 100 100 is configured to draw from about 12 mL to about 36 mL (e.g., from about 30 mL to about 33 mL, from about 33 mL to about 36 mL, from about 32 mL to about 36 mL, from about 34 mL to about 36 mL, from about 30 mL to about 32 mL, or from about 35 mL to about 36 mL, e.g., about 30 mL, about 31 mL, about 32 mL, about 33 mL, about 34 mL, about 35 mL, or about 36 mL).

[0172] In some embodiments, the volume of the PRFM layer 208 may be from about 7 mL to about 23 mL (e.g., from about 7 mL to about 12 mL, from about 10 mL to about 15 mL, from about 15 mL to about 20 mL, from about 18 mL to about 22 mL, from about 20 mL to about 23 mL, or from about 19 mL to about 21 mL, e.g., about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, or about 23 mL) when pre-filled tube 100 is configured to draw from about 14 mL to about 40 mL (e.g., from about 14 mL to about 20 mL, from about 15 mL to about 30 mL, from about 15 mL to about 25 mL, from about 25 mL to about 30 mL, from about 20 mL to about 30 mL, from about 31 mL to about 35 mL, from about 35 mL to about 38 mL, from about 38 mL to about 40 mL, from about 34 mL to about 35 mL, from about 34 mL to about 36 mL, from about 34 mL to about 38 mL, from about 36 mL to about 40 mL, e.g., about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, about 30 mL, about 31 mL, about 32 mL, about 33 mL, about 34 mL, about 35 mL, about 36 mL, about 37 mL, about 38 mL, about 39 mL, or about 40 mL of the blood sample 200).

[0173] The viscosity of the PRFM layer 208 may vary depending on the stage of coagulation. At least a portion of the PRFM layer 208 may be removed from the tube 102 with a viscosity of from about 1 cP to about 1000 cP. In some embodiments, the at least the portion of the PRFM layer 208 may be removed from the tube 102 in a fluid state, with a viscosity less than or equal to 100 cP. In some embodiments, the at least the portion of the PRFM layer 208 may be removed from the tube 102 in a gel state, with a viscosity greater than or equal to 100 cP.Attorney Docket No. 005042-00013

[0174] The PRFM layer 208 may include less than about 0.3% residual hematocrit. In some embodiments, wherein the PRFM layer 208 includes at least 2.5 x 105platelets / pL. In some embodiments, the PRFM layer 208 includes a concentration of calcium gluconate and / or calcium chloride of from about 3 mg / mL to about 20 mg / mL (e.g., about 3 mg / mL to about 6 mg / mL, about 3 mg / mL to about 12 mg / mL, about 6 mg / mL to about 12 mg / mL, about 12 mg / mL to about 20 mg / mL, about 12 mg / mL to about 15 mg / mL, about 10 mg / mL to about 15 mg / mL, about 15 mg / mL to about 20 mg / mL, e.g., about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, or about 20 mg / mL).

[0175] In some embodiments, the PRFM layer 208 may include a concentration of ascorbic acid of from about 20 mg / mL to about 75 mg / mL (e.g., about 20 mg / mL to about 30 mg / mL, about 25 mg / mL to about 50 mg / mL, about 45 mg / mL to about 55 mg / mL, about 45 mg / mL to about 65 mg / mL, about 50 mg / mL to about 60 mg / mL, about 60 mg / mL to about 65 mg / mL, about 65 mg / mL to about 75 mg / mL, e.g., about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 32 mg / mL, about 35 mg / mL, about 36 mg / mL, about 37 mg / mL, about 38 mg / mL, about 39 mg / mL, about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 47 mg / mL, about 50 mg / mL, about 52 mg / mL, about 55 mg / mL, about 57 mg / mL, about 60 mg / mL, about 61 mg / mL, about 62 mg / mL, about 63 mg / mL, about 64 mg / mL, about 65 mg / mL, about 67 mg / mL, about 70 mg / mL, about 72 mg / mL, or about 75 mg / mL).EXAMPLE 1

[0232] Table 1 : Contents of the pre-filled tube 100 configured to draw from about 2 ml to about 12 ml of the blood sample 200Attorney Docket No. 005042-00013

[0233] The tube 102 of the pre-filled tube 100 may be configured to draw from about 2 mL to about 12 mL of the blood sample 200 (e.g., about 2 mL to about 10 mL, about 2 mL to about 8 mL, about 2 mL to about 4 mL, about 4 mL to about 8 mL, about 4 mL to about 12 mL, about 8 mL to about 10 mL, about 10 mL to about 12 mL, e.g., about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, or about 12 mL of the blood sample 200) from a subject.

[0234] Table 1 describes the content of each of the five layers in the pre-filled tube 100 of the disclosure configured to draw from about 2 mL to about 12 mL of the blood sample 200, along with the size and materials of the sealing cap 130 and tube 102. The first drug layer 110 and the second drug layer 112 may each have a volume of about 0.7 mL. The first drug layer 110 may include about 100 mg / mL calcium gluconate and / or calcium chloride as a coagulating agent. The second drug layer 112 may include ascorbic acid at a concentration ofAttorney Docket No. 005042-00013about 500 mg / mL. As such, first drug layer 110 may include about 70 mg of calcium gluconate and / or calcium chloride, and the second drug layer 112 may include about 350 mg of ascorbic acid. The first gel layer 120 and the second gel layer 122 may each include a non-sterile, thixotropic separation serum gel and may each have a volume of about 0.7 mL, or, equivalently, a mass of about 0.75 g. The third drug layer 114 may be an anticoagulant including ACD-A and may have a volume of about 1.1 mL. ACD-A may include about 7 mg / mL citric acid, about 22 mg / mL sodium citrate, and about 24.5 mg / mL dextrose. With this, the third drug layer 114 may include about 9 mg of citric acid, about 24.2 mg of sodium citrate, and about 27 mg of dextrose. The tube 102 may be a clear, silicone-coated PRP tube with a size of 16 mm x 125 mm. The volume of the tube 102 may be 16 mL. The sealing cap 130 may be a butyl rubber tube stopper that has a size (e.g., diameter) of 16 mm. The pressure of the tube 102 may be about negative 95 kPa. The volume of the space 108 may be about 11 mL. The sealing cap 130 may displace about 1 mL of the volume of the tube 102. The ratio of the volume of the blood sample 200 to the combined volume of the first drug layer 110, the second drug layer 112, and the third drug layer 114 may about 4:1.EXAMPLE 2

[0235] Table 2: Contents of the pre-filled tube 100 configured to draw from about 7 mL to about 22 mL of the blood sample 200.Attorney Docket No. 005042-00013

[0236] The tube 102 of the pre-filled tube 100 may be configured to draw from about 7 mL to about 22 mL of the blood sample 200 (e.g., from about 7 mL to about 10 mL, about 10 mL to about 18 mL, about 8 mL to about 12 mL, from about 18 mL to about 20 mL, from about 19 mL to about 20 mL, from about 20 mL to about 22 mL, e.g., about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, or about 22 mL of blood) from a subject. As little as 2.0 mL of the blood sample 200 may be collected (e.g., if the vein collapses during the blood draw).

[0237] Table 2 describes the content of each of the five layers in the pre-filled tube 100 of the disclosure configured to draw from about 7 mL to about 22 mL of the blood sample 200 along with the size and materials of the sealing cap 130 and the tube 102. The first drug layer 110 and second drug layer 112 may each have a volume of about 1.3 mL. The first drug layer 110 may include about 100 mg / mL calcium gluconate and / or calcium chloride as a coagulating agent. The second drug layer 112 may include about 500 mg / mL ascorbic acid as a stabilizing agent. As such, first drug layer 110 may include about 130 mg of calcium gluconate and / or calcium chloride, and second drug layer 112 may include about 650 mg of ascorbic acid. The first gel layer 120 and the second gel layer 122 may each have a volume of about 1.9 mL, or, equivalently, a mass of about 2.0 g. The third drug layer 114 may include an anticoagulant of ACD-A and may have a volume of about 2.2 mL. ACD-A may include about 7 mg / mL citric acid, about 22 mg / mL sodium citrate, and about 24.5 mg / mL dextrose.Attorney Docket No. 005042-00013With this, the third drug layer 114 may include about 15 mg of citric acid, about 48 mg of sodium citrate, and about 54 mg of dextrose. The volume of the tube 102 may be about 32 mL. The tube 102 may be a clear, silicone-coated PRP tube with a size of 20 mm x 125 mm. The sealing cap 130 may be a butyl rubber tube stopper that has a size (e.g., diameter) of 20 mm. The pressure of the tube 102 may be about negative 95 kPa. The volume of the space 140 may be about 22 mL. The sealing cap 130 may displace about 1.9 mL of the volume of the tube 102. The ratio of the volume of the blood to the combined volume of first drug layer 110, second drug layer 112, and the third drug layer 114 is about 4: 1.EXAMPLE 3

[0238] Table 3: Contents of the pre-filled tube 100 configured to draw from about 12 mL to about 36 mL of the blood sample 200.Attorney Docket No. 005042-00013

[0239] The tube 102 of the pre-filled tube 100 may be configured to draw from about 12 mL to about 36 mL of the blood sample 200 (e.g., from about 12 mL to about 30 mL, from about 12 mL to about 20 mL, from about 15 mL to about 30 mL, from about 15 mL to about 20 mL, from about 30 mL to about 33 mL, from about 33 mL to about 36 mL, from about 32 mL to about 36 mL, from about 34 mL to about 36 mL, from about 30 mL to about 32 mL, or from about 35 mL to about 36 mL, e.g., about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, about 30 mL, about 31 mL, about 32 mL, about 33 mL, about 34 mL, about 35 mL, or about 36 mL of the blood sample 200) from a subject. As little as 2.0 mL of the blood sample 200 may be collected (e.g., if the vein collapses during the blood draw).

[0240] Table 3 describes the content of each of the five layers in the pre-filled tube 100 of the disclosure configured to draw from about 12 mL to about 36 mL of the blood sample 200, along with the size and materials of the sealing cap 130 and tube 102. The first drug layer 110 and the second drug layer 112 may each have a volume of about 2.0 mL. The first drug layer 110 may include about 100 mg / mL calcium gluconate and / or calcium chloride as a coagulating agent. The second drug layer 112 may include about 500 mg / mL ascorbic acid as a stabilizing agent. As such, first drug layer 110 may include about 200 mg of calcium gluconate and / or calcium chloride, and second drug layer 112 may include about 1000 mg of ascorbic acid. The first gel layer 120 and the second gel layer 122 may each have a volume of about 4.3 mL, or, equivalently, a mass of about 4.5 g. The third drug layer 114 may include ACD-A as an anticoagulant and may have a volume of about 3.2 mL. ACD-A may include about 7 mg / mL citric acid, about 22 mg / mL sodium citrate, and about 24.5 mg / mL dextrose. With this, the third drug layer 114 may include about 22 mg of citric acid, about 70 mg of sodium citrate, and about 78 mg of dextrose. The volume of the tube 102 may be about 54 mL. The tube 102 may be a clear, silicone-coated PRP tube with a size of 29 mm x 115 mm.Attorney Docket No. 005042-00013The sealing cap 130 may be a butyl rubber tube stopper that has a size (e.g., diameter) of 29 mm. The pressure of the tube 102 may be about negative 85 kPa. The volume of the space 140 may be about 35 mL. The sealing cap 130 may displace about 3.5 mL of the volume of the tube 102. The ratio of the volume of the blood to the combined volume of first drug layer 110, second drug layer 112, and the third drug layer 114 is about 4: 1.EXAMPLE 4

[0241] Table 4: Contents of the pre-filled tube 100 configured to draw from about 14 mL to about 40 mL of the blood sample 200 (e.g., about 31.6 mL of the blood sample 200).Attorney Docket No. 005042-00013

[0242] The tube 102 of the pre-filled tube 100 may be configured to draw from about 14 mL to about 40 mL of the blood sample 200 (e.g., from about 14 mL to about 20 mL, from about 15 mL to about 30 mL, from about 15 mL to about 25 mL, from about 25 mL to about 30 mL, from about 20 mL to about 30 mL, from about 31 mL to about 35 mL, from about 35 mL to about 38 mL, from about 38 mL to about 40 mL, from about 34 mL to about 35 mL, from about 34 mL to about 36 mL, from about 34 mL to about 38 mL, from about 36 mL to about 40 mL, e.g., about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, about 30 mL, about 31 mL, about 32 mL, about 33 mL, about 34 mL, about 35 mL, about 36 mL, about 37 mL, about 38 mL, about 39 mL, or about 40 mL of the blood sample 200 from a subject. As little as 2.0 mL of the blood sample 200 may be collected (e.g., if the vein collapses during the blood draw).

[0243] Table 4 describes the content of each of the five layers in the pre-filled tube 100 of the disclosure configured to draw from about 14 mL to about 40 mL of the blood sample 200, along with the size and materials of the sealing cap 130 and tube 102. The first drug layer 110 may include about 100 mg / mL calcium gluconate and / or calcium chloride as a coagulating agent. The second gel layer may include about 500 mg / mL ascorbic acid as a stabilizing agent. The first drug layer 110 and the second drug layer 112 may each have a volume of about 2.2 mL. As such, first drug layer 110 may include about 220 mg of calcium gluconate and / or calcium chloride, and second drug layer 112 may include about 1220 mg of ascorbic acid. The first gel layer 120 and the second gel layer 122 may each have a volume of about 4.3 mL, or, equivalently, a mass of about 4.5 g. The third drug layer 114 may include ACD-A as an anticoagulant and may have a volume of about 3.6 mL. ACD-A may include about 7 mg / mL citric acid, about 22 mg / mL sodium citrate, and about 24.5 mg / mL dextrose. With this, the third drug layer 114 may include about 25 mg of citric acid, about 79 mg of sodium citrate, and about 88 mg of dextrose. The volume of the tube 102 may be about 54 mL. The tube 102 may be a clear, silicone-coated PRP tube with a size of 29 mm x 115 mm. The sealing cap 130 may be a butyl rubber tube stopper that has a size (e.g., diameter) of 29 mm. The pressure of the tube 102 may be about negative 95 kPa. The volume of the space 140 may be about 34 mL. The sealing cap 130 may displace about 3.5 mL of the volume of the tube 102. The ratio of the volume of the blood sample 200 to the combined volume of first drug layer 110, second drug layer 112, and the third drug layer 114 is about 4:1.Attorney Docket No. 005042-00013Example 5

[0244] Table 5 Contents of the PRFM preparation tube 100’ configured to draw from about 2 mL to about 12 mL of the blood sample 200.

[0245] The tube 102 of the PRFM preparation tube 100’ may be configured to draw from about 2 mL to about 12 mL of blood (e.g., about 2 mL to about 10 mL, about 2 mL to about 8 mL, about 2 mL to about 4 mL, about 4 mL to about 8 mL, about 4 mL to about 12 mL, about 8 mL to about 10 mL, about 10 mL to about 12 mL, e.g., about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, or about 12 mL of blood) from a subject.

[0246] Table 5 describes the content of each of the three layers in the pre-filled tube 100’ of the disclosure configured to draw from about 2 mL to about 12 mL of blood, along with the size and materials of sealing cap 130 and tube 102. The first drug layer 110 and the second drug layer 112 can each have a volume of about 0.7 mL. The first drug layer 110 can include calcium gluconate as a coagulating agent and / or calcium chloride at a concentration of about 100 mg / mL. The second drug layer 112 can include ascorbic acid as a coagulating agent at aAttorney Docket No. 005042-00013concentration of about 500 mg / mL. As such, first drug layer 110 can include about 70 mg of calcium gluconate and / or calcium chloride, and second drug layer 112 can include about 350 mg of ascorbic acid. The gel layer 120 can include a non-sterile, thixotropic separation serum gel and can have a volume of about 1.4 mL, or, equivalently, a mass of about 1.5g. The volume of tube 102 can be about 16 mL. Tube 102 can be a clear, silicone-coated PRP tube with a size of 16 mm by 125 mm. Sealing cap 130 can be a butyl rubber tube stopper that has a size (e.g., diameter) of 16 mm. The pressure of tube 102 can be about negative 95 kPa. The volume of space 108 may be about 12 mL. The sealing cap 130 may displace about 1 mL of the volume of tube 102. The ratio of the volume of the blood sample 200 to the combined volume of first drug layer 110 and second drug layer 112 is about 8:1.EXAMPLE 6

[0247] Table 6: Contents of the pre-filled tube 100 configured to draw from about 7 mL to about 22 mL of the blood sample 200.Attorney Docket No. 005042-00013

[0248] The tube 102 of the pre-filled tube 100’ may be configured to draw from about 7 mL to about 22 mL of the blood sample 200 (e.g., from about 7 mL to about 10 mL, about 10 mL to about 18 mL, about 8 mL to about 12 mL, from about 18 mL to about 20 mL, from about 19 mL to about 20 mL, from about 20 mL to about 22 mL, e.g., about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, or about 22 mL of the blood sample 200) from a subject. As little as 2.0 mL of the blood sample 200 may be collected (e.g., if the vein collapses during the blood draw).

[0249] Table 6 describes the content of each of the three layers in the pre-filled tube 100’ of the disclosure configured to draw from about 7 mL to about 22 mL of the blood sample 200, along with the size and materials of the sealing cap 130 and tube 102. First drug layer 110 and second drug layer 112 may each have a volume of about 1.3 mL. First drug layer 110 may include calcium gluconate and / or calcium chloride as a coagulating agent at a concentration of about 100 mg / mL. The second drug layer 112 may include ascorbic acid as a stabilizing agent at a concentration of about 500 mg / mL. As such, the first drug layer 110 may include about 130 mg of calcium gluconate and / or calcium chloride, and the second drug layer 112 may include about 650 mg of ascorbic acid. The gel layer 120 may include a non-sterile, thixotropic separation serum gel and may have a volume of about 4 mL, or, equivalently, a mass of about 3.8 g. The volume of the tube 102 may be about 32 mL. The tube 102 may be a clear, silicone-coated PRP tube with a size of 20 x 125 mm. Sealing cap 130 may be a butyl rubber tube stopper that has a size (e.g., diameter) of 20 mm. The pressure of the tube 102 may be about negative 95 kPa. The volume of the space 108 may be about 24 mL. The sealing cap 130 may displace about 1.9 mL of the volume of the tube 102. The ratio of the volume of the blood to the combined volume of first drug layer 110 and second drug layer 112 is about 8:1.EXAMPLE 7

[0250] Table 7: Contents of the pre-filled tube 100’ configured to draw from about 12 mL to about 36 mL of blood.Attorney Docket No. 005042-00013

[0251] The tube 102 of the pre-filled tube 100’ may be configured to draw from about 12 mL to about 36 mL of blood (e.g., from about 12 mL to about 30 mL, from about 12 mL to about 20 mL, from about 15 mL to about 30 mL, from about 15 mL to about 20 mL, from about 30 mL to about 33 mL, from about 33 mL to about 36 mL, from about 32 mL to about 36 mL, from about 34 mL to about 36 mL, from about 30 mL to about 32 mL, or from about 35 mL to about 36 mL, e.g., about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, about 30 mL, about 31 mL, about 32 mL, about 33 mL, about 34 mL, about 35 mL, or about 36 mL of blood) from a subject. As little as 2.0 mL of blood may be collected (e.g., if the vein collapses during the blood draw).

[0252] Table 7 describes the content of each of the three layers in the pre-filled tube 100’ of the disclosure configured to draw from about 12 mL to about 36 mL of blood, along with the size and materials of the sealing cap 130 and tube 102. The first drug layer 110 and theAttorney Docket No. 005042-00013second drug layer 112 may each have a volume of about 2 mL. The first drug layer 110 may include calcium gluconate and / or calcium chloride as a coagulating agent at a concentration of about 100 mg / mL. The second drug layer 112 may include ascorbic acid as a stabilizing agent at a concentration of about 500 mg / mL. As such, the first drug layer 110 may include about 200 mg of calcium gluconate and / or calcium chloride, and second drug layer 112 may include about 1000 mg of ascorbic acid. The gel layer 120 may include a non-sterile, thixotropic separation serum gel and may have a volume of about 8.5 mL, or, equivalently, a mass of about 9 g. The volume of the tube 102 may be about 54 mL. The tube 102 may be a clear, silicone-coated PRP tube with a size of 29 x 115 mm. The sealing cap 130 may be a butyl rubber tube stopper that has a size (e.g., diameter) of 29 mm. The pressure of the tube 102 may be about negative 85 kPa. The volume of the space 108 may be about 38 mL. The sealing cap 130 may displace about 3.5 mL of the volume of the tube 102. The ratio of the volume of the blood to the combined volume of first drug layer 110 and the second drug layer 112 is about 8:1.EXAMPLE 8

[0253] Table 8. Contents of the pre-filled tube 100’ configured to draw from about 14 mL to about 40 mL of blood.Attorney Docket No. 005042-00013

[0254] The tube 102 of the pre-filled tube 100’ may be configured to draw from about 14 mL to about 40 mL of blood (e.g., from about 14 mL to about 20 mL, from about 15 mL to about 30 mL, from about 15 mL to about 25 mL, from about 25 mL to about 30 mL, from about 20 mL to about 30 mL, from about 31 mL to about 35 mL, from about 35 mL to about 38 mL, from about 38 mL to about 40 mL, from about 34 mL to about 35 mL, from about 34 mL to about 36 mL, from about 34 mL to about 38 mL, from about 36 mL to about 40 mL, e.g., about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, about 30 mL, about 31 mL, about 32 mL, about 33 mL, about 34 mL, about 35 mL, about 36 mL, about 37 mL, about 38 mL, about 39 mL, or about 40 mL of blood) from a subject. As little as 2.0 mL of blood may be collected (e.g., if the vein collapses during the blood draw).

[0255] Table 8 describes the content of each of the three layers in the pre-filled tube 100’ of the disclosure configured to draw from about 14 mL to about 40 mL of blood, along with the size and materials of the sealing cap 130 and tube 102. The first drug layer 110 and the second drug layer 112 may each have a volume of about 2.2 mL. The first drug layer 110 may include calcium gluconate and / or calcium chloride as a coagulating agent at a concentration of about 100 mg / mL. The second drug layer 112 may include ascorbic acid as a stabilizing agent at a concentration of about 500 mg / mL. As such, the first drug layer 110 may include 220 mg of calcium gluconate and / or calcium chloride, and the second drug layer 112 may include 1,100 mg of ascorbic acid. The gel layer 120 may include a non-sterile, thixotropic separation serum gel and may have a volume of about 8.5 mL, or, equivalently, a mass of about 9 g. The volume of the tube 102 may be about 54 mL. The tube 102 may be a clear, silicone-coated PRP tube with a size of 29 x 115 mm. The sealing cap 130 may be a butyl rubber tube stopper that has a size (e.g., diameter) of 29 mm. The pressure of the tube 102 may be about negative 95 kPa. The volume of the space 108 may be about 38 mL. TheAttorney Docket No. 005042-00013sealing cap 130 may displace about 3.5 mL of the volume of the tube 102. The ratio of the volume of the blood to the combined volume of first drug layer 110 and second drug layer 112 is about 8:1.EXAMPLE 9

[0256] Table 9. Contents of the pre-filled tube 100’ configured to draw from about 2 mL to about 12 mL of blood.

[0257] The tube 102 of the pre-filled tube 100’ may be configured to draw from about 2 mL to about 12 mL of blood (e.g., about 2 mL to about 10 mL, about 2 mL to about 8 mL, about 2 mL to about 4 mL, about 4 mL to about 8 mL, about 4 mL to about 12 mL, about 8 mL to about 10 mL, about 10 mL to about 12 mL, e.g., about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, or about 12 mL of blood) from a subject. As little as 2.0 mL of blood may be collected (e.g., if the vein collapses during the blood draw).Attorney Docket No. 005042-00013

[0258] Table 9 describes the content of each of the three layers in the pre-filled tube 100’ of the disclosure configured to draw from about 2 mL to about 12 mL of blood, along with the size and materials of the sealing cap 130 and the tube 102. The first drug layer 110 may include calcium gluconate and / or calcium chloride as a coagulating agent at a concentration of 100 mg / mL and may have a volume of about 0.7 mL. As such, the first drug layer 110 may include about 70 mg of calcium gluconate and / or calcium chloride. The gel layer 120 may include a non-sterile, thixotropic separation serum gel and may have a volume of about 1.4 mL, or, equivalently, a mass of about 1.5 g. The second drug layer 112 may include ACD-A as an anticoagulant and may have a volume of about 1.1 mL. ACD-A may include about 7 mg / mL citric acid, about 22 mg / mL sodium citrate, and about 24.5 mg / mL dextrose. With this, the second drug layer 112 may include about 8 mg of citric acid, about 24 mg of sodium citrate, and about 27 mg of dextrose. The volume of the tube 102 may be about 16 mL. The tube 102 may be a clear, silicone-coated PRP tube with a size of 16 x 125 mm. Sealing cap 130 may be a butyl rubber tube stopper that has a size (e.g., diameter) of 16 mm. The pressure of the tube 102 may be about negative 95 kPa. The volume of the space 108 may be about 12 mL. Sealing cap 130 may displace about 1 mL of the volume of the tube 102. The ratio of the volume of the blood to the combined volume of the first drug layer 110 and the second drug layer 112 is about 6:1.EXAMPLE 10

[0259] Table 10. Contents of the pre-filled tube 100’ configured to draw from about 7 mL to about 22 mL of blood.Attorney Docket No. 005042-00013

[0260] The tube 102 of the pre-filled tube 100’ may be configured to draw from about 7 mL to about 22 mL of blood (e.g., from about 7 mL to about 10 mL, about 10 mL to about 18 mL, about 8 mL to about 12 mL, from about 18 mL to about 20 mL, from about 19 mL to about 20 mL, from about 20 mL to about 22 mL, e.g., about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, or about 22 mL of blood) from a subject. As little as 2.0 mL of blood may be collected (e.g., if the vein collapses during the blood draw).

[0261] Table 10 describes the content of each of the three layers in the pre-filled tube 100’ of the disclosure configured to draw from about 7 mL to about 22 mL of blood, along with the size and materials of the sealing cap 130 and tube 102. The first drug layer 110 may include calcium gluconate and / or calcium chloride as a coagulating agent at a concentration of 100 mg / mL and may have a volume of about 1.3 mL. As such, the first drug layer 110 may include about 130 mg of calcium gluconate and / or calcium chloride. The gel layer 120 may include a non-sterile, thixotropic separation serum gel and may have a volume of about 3.8 mL, or, equivalently, a mass of about 4 g. The second drug layer 112 may include ACD-A as an anticoagulant and may have a volume of about 2.2 mL. ACD-A may include about 7 mg / mL citric acid, about 22 mg / mL sodium citrate, and about 24.5 mg / mL dextrose. With this, the second drug layer 112 may include about 15 mg of citric acid, about 48 mg of sodium citrate, and about 54 mg of dextrose. The volume of the tube 102 may be about 32 mL. The tube 102 may be a clear, silicone-coated PRP tube with a size of 20 x 125 mm. The sealing cap 130 may be a butyl rubber tube stopper that has a size (e.g., diameter) of 20 mm. The pressure of the tube 102 may be about negative 95 kPa. The volume of the space 108Attorney Docket No. 005042-00013may be about 23 mL. The sealing cap 130 may displace about 1.9 mL of the volume of the tube 102. The ratio of the volume of the blood to the combined volume of drug layer 110 and the second drug layer 112 is about 6:1.EXAMPLE 11

[0262] Table 11 Contents of the pre-filled tube 100’ configured to draw from about 12 mL to about 36 mL of blood.

[0263] The tube 102 of the pre-filled tube 100’ may be configured to draw from about 12 mL to about 36 mL of blood (e.g., from about 12 mL to about 30 mL, from about 12 mL to about 20 mL, from about 15 mL to about 30 mL, from about 15 mL to about 20 mL, from about 30 mL to about 33 mL, from about 33 mL to about 36 mL, from about 32 mL to about 36 mL, from about 34 mL to about 36 mL, from about 30 mL to about 32 mL, or from about 35 mL to about 36 mL, e.g., about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23Attorney Docket No. 005042-00013mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, about 30 mL, about 31 mL, about 32 mL, about 33 mL, about 34 mL, about 35 mL, or about 36 mL of blood) from a subject. As little as 2.0 mL of blood may be collected (e.g., if the vein collapses during the blood draw).

[0264] Table 11 describes the content of each of the three layers in the pre-filled tube 100’ of the disclosure configured to draw from about 12 mL to about 36 mL of blood, along with the size and materials of the sealing cap 130 and tube 102. The first drug layer 110 may include calcium gluconate and / or calcium chloride as a coagulating agent at a concentration of about 100 mg / mL and may have a volume of about 2 mL. As such, the first drug layer 110 may include about 200 mg of calcium gluconate and / or calcium chloride. The gel layer 120 may include a non-sterile, thixotropic separation serum gel and may have a volume of about 8.6 mL, or, equivalently, a mass of about 9 g. The second drug layer 112 may include ACD-A as an anticoagulant and may have a volume of about 3.2 mL. ACD-A may include about 7 mg / mL citric acid, about 22 mg / mL sodium citrate, and about 24.5 mg / mL dextrose. With this, the second drug layer 112 may include about 22 mg of citric acid, about 70 mg of sodium citrate, and about 78 mg of dextrose. The volume of the tube 102 may be about 54 mL. The tube 102 may be a clear, silicone-coated PRP tube with a size of 29 x 115 mm. The sealing cap 130 may be a butyl rubber tube stopper that has a size (e.g., diameter) of 29 mm. The pressure of the tube 102 may be about negative 85 kPa. The volume of the space 108 may be about 37 mL. The sealing cap 130 may displace about 3.5 mL of the volume of the tube 102. The ratio of the volume of the blood to the combined volume of drug layer 110 and the second drug layer 112 is about 6:1.EXAMPLE 12

[0265] Table 12 Contents of the pre-filled tube 100’ configured to draw from about 14 mL to about 40 mL of blood.Attorney Docket No. 005042-00013

[0266] The tube 102 of the pre-filled tube 100’ may be configured to draw from about 14 mL to about 40 mL of blood (e.g., from about 14 mL to about 20 mL, from about 15 mL to about 30 mL, from about 15 mL to about 25 mL, from about 25 mL to about 30 mL, from about 20 mL to about 30 mL, from about 31 mL to about 35 mL, from about 35 mL to about 38 mL, from about 38 mL to about 40 mL, from about 34 mL to about 35 mL, from about 34 mL to about 36 mL, from about 34 mL to about 38 mL, from about 36 mL to about 40 mL, e.g., about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, about 30 mL, about 31 mL, about 32 mL, about 33 mL, about 34 mL, about 35 mL, about 36 mL, about 37 mL, about 38 mL, about 39 mL, or about 40 mL of the blood sample 200) from a subject. As little as 2.0 mL of the blood sample 200 may be collected (e.g., if the vein collapses during the blood draw).

[0267] Table 12 describes the content of each of the three layers in the pre-filled tube 100’ of the disclosure configured to draw from about 14 mL to about 40 mL of the blood sample 200, along with the size and materials of the sealing cap 130 and tube 102. The first drug layer 110 may include calcium gluconate and / or calcium chloride as a coagulating agent at a concentration of about 100 mg / mL and may have a volume of about 2.2 mL. As such, the first drug layer 110 may include about 220 mg of calcium gluconate and / or calcium chloride. The gel layer 120 may include a non-sterile, thixotropic separation serum gel and may have a volume of about 8.5 mL, or, equivalently, a mass of about 9 g. The third drug layer 112 mayAttorney Docket No. 005042-00013include ACD-A as an anticoagulant and may have a volume of about 3.6 mL. ACD-A may include about 7 mg / mL citric acid, about 22 mg / mL sodium citrate, and about 24.5 mg / mL dextrose. With this, the third drug layer 114 may include about 25 mg of citric acid, about 79 mg of sodium citrate, and about 88 mg of dextrose. The volume of the tube 102 may be about 54 mL. Tube 102 may be a clear, silicone-coated PRP tube with a size of 29 x 115 mm. The sealing cap 130 may be a butyl rubber tube stopper that has a size (e.g., diameter) of 29 mm. The pressure of the tube 102 may be about negative 95 kPa. The volume of the space 108 may be about 37 mL. The sealing cap 130 may displace about 3.5 mL of the volume of the tube 102. The ratio of the volume of the blood to the combined volume of the first drug layer 110 and the second drug layer 112 is about 6:1.WOUND DRESSING

[0268] FIGS. 4-14 illustrate various views of a wound dressing 1000 of the present disclosure. As illustrated, the wound dressing 1000 may include a bandage 1002. The bandage 1002 may be applied to a subject (e.g., to skin) to retain the biomaterial in place against a wound while allowing gas (e.g., air) to pass therethrough. In some embodiments, an injection port 1100 may be attached to a distal surface of the bandage 1002 and be configured to allow passage of the biomaterial through the bandage 1002. The bandage 1002 may be flexible and / or conformable. In some embodiments, the bandage 1002 may be configured to retain a shape when bent. The bandage 1002 may conform to the application site of the subject, inhibiting leakage, and / or promoting wearability and comfort.

[0269] As illustrated in FIG. 6, the bandage 1002 may include a first layer 1020 and a second layer 1040. The first layer 1020 may have a distal surface 1022 and a proximal surface 1024, and the second layer 1040 may have a distal surface 1042 and a proximal surface 1044. The second layer 1040 may at least partially (e.g., fully) cover the first layer 1020, such that the first layer 1020 may be positioned between the proximal surface 1044 of the second layer 1040 and the skin of the subject. The proximal surface 1024 of the first layer 1020 may be at least partially positioned against the wound, and / or the distal surface 1042 of the second layer 1040 may be at least partially uncovered and directly open or exposed to the atmospheric environment.Attorney Docket No. 005042-00013

[0270] In some embodiments, the second layer 1040 may be a substantially planar sheet, as illustrated in FIG. 6. In some embodiments, the second layer 1040 may be contoured to provide a cavity, for example as illustrated in FIGS. 19 and 20.

[0271] As further illustrated in FIG. 6, the first layer 1020 may be a porous material, including a plurality of first pores 1030. The first layer 1020 may be a single layer. The first layer 1020 may be a mesh defining the first pores 1030. The first layer 1020 may be nonwoven and / or non-absorbent to prevent retention of biological fluids and / or clogging of the first pores 1030. The mesh may be made polyethylene (PE), polypropylene (PP), polyester (PET), nylon (polyamide), polytetrafluoroethylene (PTFE), high-density polyethylene (HDPE), and / or polyvinylidene fluoride (PVDF).

[0272] The first pores 1030 may be sized for venting gas (e.g., air) from the area proximal of the wound dressing 1000. For example, the first pores 1030 may vent gas during injection to release gas displaced by the biomaterial directly to the atmosphere. The first pores 1030 may also allow venting once the biomaterial has been delivered to promote the drying and / or transitioning of the biomaterial to a gel state, thereby forming a gel on the skin of the subject. In some embodiments, the first pores 1030 may be liquid permeable (e.g., blood or wound exudate) but prevent passage of the biomaterial to retain the biomaterial against the wound. The first pores 1030 may have a first width of from about 0.2 microns to about 500 microns, inclusive. In some embodiments, the first width may be from about 50 microns to about 400 microns, inclusive. In some embodiments, the first width may be from about 100 microns to about 400 microns, inclusive. In some embodiments, the first width may be from about 100 microns to about 300 microns, inclusive. In some embodiments, the first width may be from about 100 microns to about 200 microns, inclusive. In some embodiments, the first width may be about 150 microns. The first layer 1020 may have at least 100 of the first pores 1030. In some embodiments, the first layer 1020 may have at least 1000 of the first pores 1030. The first pores 1030 may extend through substantially the entire surface area of the first layer 1020 forming the mesh.

[0273] The second layer 1040 may include one or more (e.g., a plurality of) second pores 1050. The second layer 1040 may be non-woven and / or non-absorbent to prevent retention of biological fluids and / or clogging of the second pores 1050. The second layer 1040 may be made of polyvinyl chloride (PVC), polyethylene (PE), and / or thermoplastic polyurethane (TPU). The second layer 1040 may be a sheet (e.g., a plastic sheet) or film (e.g., plastic film)Attorney Docket No. 005042-00013sized to cover (e.g., fully or partially cover) the first layer 1020. The second layer 1040 may be an external cover or surface of the bandage 1002 that is at least partially uncovered by any another layer. The second pores 1050 may be directly open or exposed to the atmospheric environment, for example, without any suction or negative pressure being applied against the second layer 1040.

[0274] The second pores 1050 may be sized for venting gas (e.g., air), biomaterial (e.g., PRP or PRFM), and / or a biological fluid (e.g., blood or wound exudate) from proximal of the wound dressing 1000 to the atmosphere (e.g., at atmospheric pressure). In some embodiments, the second pores 1050 may have a shape selected from a circle, a square, a star, an oval, a rectangle, a triangle, a polygon, or any combination thereof. The second pores 1050 may have a second width from about 1 mm to about 10 mm, inclusive. In some embodiments, the second pores 1050 may have a width from about 1 mm to about 8 mm, inclusive. In some embodiments, the second pores 1050 may have a width from about 1 mm to about 4 mm, inclusive. In some embodiments, the second pores 1050 may have a width from about 1 mm to about 2 mm, inclusive. In some embodiments, the second pores 1050 may have a width from about 2 mm to about 4 mm, inclusive. The second layer 1040 may have less than one thousand of the second pores 1050. In some embodiments, the second layer 1040 may have less than one hundred of the second pores 1050. For example, as illustrated, the second layer 1040 may have less than fifty of the second pores 1050. It is also contemplated that the one or more second pores 1050 may consist of a single enlarged opening covering at least a portion of the first layer 1020.

[0275] The wound dressing 1000 may be used at atmospheric pressure, without a source of suction or vacuum. Thus, the distal surface 1042 may be at least partially uncovered and exposed to the atmospheric environment. For example, the distal surface 1042 may be uncovered except for a portion covered by the injection port 1100. The second pores 1050 may be uncovered distally with direct exposure to atmospheric air to allow the wound dressing 1000 to vent gases (e.g., air) from the wound site through the first layer 1020 and the second layer 1040, and directly to the atmospheric environment without any source of suction or vacuum. Thus, the wound dressing 1000 may allow for pressure equalization during injection of the biomaterial and promote coagulation of the biomaterial over an extended period of time without the inconvenience of external equipment.Attorney Docket No. 005042-00013

[0276] The second layer 1040 may retain the first layer 1020 against the wound while retaining porosity of the first layer 1020. The second layer 1040 may have a second perimeter that is larger than a first perimeter of the first layer 1020. The second layer 1040 may have a second surface area that is larger than a first surface area of the first layer 1020. The second pores 1050 may be on a central portion 1046 of the second layer 1040 overlying the first layer 1020. The central portion 1046 of the second layer 1040 overlying the first layer 1020 may have a second porosity greater than a first porosity of the first layer 1020. The second width of the second pores 1050 may be greater than the first width of the first pores 1030. The first pores 1030 may be more densely arranged than the second pores. The number of first pores 1030 in the first layer 1020 may be larger than the number of second pores 1050 in the second layer 1040.

[0277] As illustrated in FIG. 4, the central portion 1046 of the second layer 1040 overlying the first layer 1020 may be centrally disposed along the surface area of the second layer 1040 (e.g., centrally disposed along an X-axis and / or a Y-axis of the second layer 1040). A perimeter portion 1048 may have a width extending at least partially around the central portion 1046. For example, as illustrated, the central portion 1046 may be centrally disposed along the X-axis and the Y-axis, forming a perimeter portion 1048 around the entire perimeter of the second layer 1040. The injection port 1110 may be adhered to the distal surface 1042 of the perimeter portion 1048.

[0278] An adhesive 1080 may be applied on the proximal surface 1044 of the perimeter portion 1048. The adhesive 1080 on the perimeter portion 1048 may adhere the bandage 1002 against the subject (e.g., the skin). In some embodiments, the adhesive 1080 may be applied to the entire proximal surface 1044 of the second layer 1040. When applied to the entire proximal surface 1044, the adhesive 1080 may adhere the first layer 1020 to the second layer 1040. The adhesive 1080 may adhere the first layer 1020 to the second layer 1040, for example by penetrating the first pores 1030. At least some of the porosity of the first layer 1020 may be retained by at least a subset of the first pores 1030 that underly the second pores 1050. The second pores 1050 may not retain the adhesive 1080 (due to the lack of a surface), thus the adhesive 1080 would not penetrate the subset of first pores 1030 underlying the second pores 1050. Thus, the second pores 1050 may be at least partially aligned with the subset of first pores 1030 to allow for ventilation through the first layer 1020 and the second layer 1040. The adhesive 1080 may comprise a low-tack adhesive to enable removal of theAttorney Docket No. 005042-00013second layer 1040 from the subject. The adhesive 1080 may comprise a silicon adhesive. In some embodiments, the adhesive 1080 may be a silicon gel adhesive. The second layer 1040 may include a removable tab (not shown) configured to be peeled for removal of the bandage 1002.

[0279] In some embodiments, the adhesive 1080 may be applied only on the proximal surface 1044 of the perimeter portion 1048 to be applied to the subject. A second adhesive may adhere the first layer 1020 to the second layer 1040. The second adhesive (when present) may be a medical adhesive, such as an epoxy or an acrylic.

[0280] As illustrated in FIGS. 6-8, a liner 1200 may at least partially cover the proximal surface of the bandage 1002 before use. The liner 1200 may be a sheet (e.g., a plastic sheet) or film (e.g., plastic film) sized to cover the wound dressing 1000. When in place, the liner 1200 may cover the adhesive 1080, protect the bandage 1002, and / or prevent contamination of the first layer 1020 and / or second layer 1040. The liner 1200 may be a split liner. The liner 1200 may have a first liner portion 1202 and a second liner portion 1204 extending over the proximal surface. An overlap 1206 of the first liner portion 1202 and the second liner portion 1204 may have an adhesive to releasably secure the first liner portion 1202 and the second liner portion 1204. In some embodiments, the liner 1200 may have a full split, thereby providing complete exposure of the adhesive when removed (as illustrated in FIG. 9). In some embodiments, the liner 1200 may have a partial score, thereby providing partial exposure of the adhesive 1080. As illustrated in FIG. 9, the liner 1200 may be removed to expose the adhesive 1080 on the proximal surface 1044 of the second layer 1040, and the bandage 1002 may be affixed to the subject (e.g., over a wound to be treated).

[0281] The wound dressing 1000 may be configured to be placed on the subject (e.g., over a wound to be treated) before the biomaterial is applied. The bandage 1002 may have one or more openings 1032, 1052 therethrough configured to allow passage of the biomaterial. For example, the first layer 1020 may have a first opening 1032, and the second layer 1040 may have a second opening 1052. The first opening 1032 and the second opening 1052 may be at least partially aligned to allow passage of the biomaterial through the bandage 1002.

[0282] The bandage 1002 may have a valve 1060 configured to selectively provide passage of the biomaterial through the first opening 1032 and / or the second opening 1052. As illustrated in FIG. 6, the valve 1060 may be configured to extend over and close the secondAttorney Docket No. 005042-00013opening 1052. The valve 1060 may provide for one-way passage of the biomaterial through the bandage 1002 while preventing reverse passage or backflow. The valve 1060 may be selfsealing and seal upon removal of fluid pressure and / or the injection port 1100. The valve 1060 may be a flap valve, a duck valve, an umbrella valve, a resealable closure, a septum, and / or a pierceable membrane. The valve 1060 may be attached (e.g., integrally formed, welded, and / or adhered) to the first layer 1020 and / or the second layer 1040. In some embodiments, the valve 1060 may be a flap valve formed integrally from the second layer 1040. For example, the valve 1060 may be formed (at the same time as the second opening 1052) by cutting a curved slit through the second layer 1040 (such as a C-shaped slit as illustrated in FIG. 6) allowing the valve 1060 to pivot relative to the second opening 1050 (and the distal surface 1042) of the second layer 1040 to open the second opening 1050. Thus, the valve 1060 may be monolithic with the second layer 1040. The valve 1060 may be low profile requiring minimal clearance and be easy to manufacture. Additionally or alternatively, the valve (or a second valve) 1060 may be on the first layer 1020 to selectively open the first opening 1032, in a similar manner.

[0283] The valve 1060 may be configured to deflect relative to the first layer 1020 and / or the second layer 1040 to an open configuration allowing passage of the biomaterial through the first opening 1032 and / or the second opening 1052. The valve 1060 may be opened by applying fluid pressure and / or a physical force. The valve 1060 may be configured to deflect or pivot proximally to allow the biomaterial to pass through the first opening 1032 and / or the second opening 1052. Additionally or alternatively, the valve 1060 may be configured to open with physical pressure applied by a solid object, such as a needle or cannula of a syringe and / or a protrusion (e.g., 1121) of the injection port 1100. In some embodiments, the valve 1060 may be configured to be punctured and / or penetrated to be opened and self-seal to close.

[0284] As further illustrated, the valve 1060 may be configured to deflect distally to close the second opening 1052 preventing backflow of the biomaterial. The valve 1060 may be biased to revert back to the closed configuration to prevent backflow of the biomaterial through the first opening 1032 and / or the second opening 1052. For example, the natural resiliency of the material of the second layer 1040 may revert the valve 1060 distally to the closed configuration. A stop 1062 may be attached to the second layer 1040 to make the valve 1060 one-way. The stop 1062 may be attached (e.g., welded, adhered, and / or integrally formed) toAttorney Docket No. 005042-00013the distal surface 1042 of the second layer 1040 and overlap with the valve 1060 to block further distal movement or pivoting of the valve 1060 past the distal surface 1042. The stop 1062 may be a collar that extends at least partially around the opening 1052 to contact the valve 1060.

[0285] The biomaterial may be viscous, gelled, and / or solidified when injected. The first opening 1032 and the second opening 1052 may have a width larger than at least the first pores 1030 to allow passage of the biomaterial through the first opening 1032 and the second opening 1052, while the biomaterial is prevented from passing through the first pores 1030. In some embodiments, the first opening 1032 and / or the second opening 1052 may have a width larger than the second pores 1050. The first opening 1032 may have a width greater than a width of the second opening 1052 and / or the valve 1060 to provide clearance to allow the valve 1060 to pivot proximally and open the second opening 1052. In some embodiments, the second opening 1052 and / or valve 1060 may have a width from about 1 mm to about 6 mm, inclusive. In some embodiments, the first opening 1032 and the valve 1060 may be about 3 mm. The first opening 1032 may have a width greater than the width of the second opening 1052 and / or valve 1060 to give the valve 1060 clearance to pivot proximally. The stop 1062 may have an opening with a width slightly smaller than the width of the second opening 1052 and / or valve 1060 to block the valve 1060 from pivoting distally (e.g., past the distal surface 1042) and to prevent backflow.

[0286] The injection port 1100 may be attached to the distal surface 1042 of the second layer 1040. The injection port 1100 may be configured to introduce the biomaterial through the bandage 1002. As illustrated in FIG. 10, the injection port 1100 may include a first connector 1120, a tube 1140, and a second connector 1160. At least a portion of the injection port 1100 may be removable from the bandage 1002 after the biomaterial is introduced into the wound. In some embodiments, a portion of the injection port 1100 may be removed. For example, the tube 1140 may be releasably attached to the first connector 1120 for the tube 1140 to be removed while the first connector 1120 remains on the bandage 1002. In some embodiments, the injection port 1100 may be removed from the bandage 1002 in its entirety to increase comfort and convenience to the subject after the biomaterial is injected.

[0287] As illustrated in FIG. 11, the first connector 1120 may have an outlet 1122 at a first end portion. The outlet 1122 may be aligned with the first opening 1032 and / or the second opening 1052 for passage of the biomaterial through the bandage 1002. In someAttorney Docket No. 005042-00013embodiments, the first connector 1120 may extend through the first opening 1032 and / or the second opening 1052 to prop open the valve 1060 of the bandage 1002 when in place and close the valve 1060 when the first connector 1120 is removed from the second layer 1040. For example, the first connector 1120 may have a protrusion 1121 extending proximally from and / or past a proximal surface 123 of the first connector 1120. The protrusion 1121 may extend at least partially (e.g., entirely) around the outlet 1122. In some embodiments, the protrusion 1121 may prop open the valve 1060 when positioned on the second layer 1040 to at least partially open the second opening 1052. In some embodiments, the first connector 1120 (e.g, the projection 1121) may not physically prop open the valve 1060 and be configured to open the valve 1060 of the bandage 1002 with fluid pressure.

[0288] As further illustrated in FIG. 11, the first connector 1120 may include a channel 1124 in fluid communication with the outlet 1122. The channel 1124 may extend along the distal surface 1042 of the second layer 1040. The outlet 1112 may extend substantially perpendicular to the channel 1124 (e.g., at a 90° bend) to direct the biomaterial from the channel 1124 through the bandage 1002. The first connector 1120 may include a valve (not shown) in the outlet 1122 and / or the channel 1124 configured to prevent backflow of the biomaterial and / or provide redundancy to the valve 1060.

[0289] As illustrated in FIG. 10, the tube 1140 may connect the first connector 1120 and the second connector 1160. The tube 1140 may spatially separate the second connector 1160 from the first connector 1120 to facilitate handling and injection of the biomaterial without disturbing the bandage 1102. The tube 1140 may be flexible to prevent forces to be applied to the bandage 1102 when a container (e.g., a syringe) of the biomaterial is attached to the second connector 1160. The tube 1140 may be received in a counterbore 1126 at a second end portion of the first connector 1120. The tube 1140 may be permanently attached in the counterbore 1126 when the injection port 1100 is configured to be removed in its entirety. In some embodiments, the tube 1140 may be releasably retained in the counterbore 1126 when the tube is removed separately from the first connector 1120. The tube 1140 may be longitudinally aligned with the channel 1124 of the first connector 1120 and a channel (not shown) of the second channel 1160.

[0290] The second connector 1160 may provide access for the biomaterial to be passed through the tube 1140, the first connector 1120, and the bandage 1002. The second connector 1160 may receive an end of the tube 1140 in a first end portion and have an inlet port 1162 onAttorney Docket No. 005042-00013a second end portion configured to connect to the container of the biomaterial. The inlet port 1162 may have an opening and a connection, such as a Luer fitting (e.g., Luer slip or Luer lock), to fluidly couple to the container without leakage. The inlet port 1162 may further include an internal valve (not shown, e.g., a Luer-activated valve) configured to inhibit backflow of the biomaterial out of the inlet port 1162. The inlet port 1162 may be configured to provide access through the bandage 1002 after application of the wound dressing 1000 to the subject. The inlet port 1162 may have an opening with a width from about 0.5 mm to about 10 mm.

[0291] As further illustrated in FIGS. 12-14, the first connector 1120 may have a tubular portion 1128 defining the channel 1124, a flange 1130 at a first end portion of the tubular portion 1128, and a tab 1132 at a second end portion of the tubular portion 1128. The first connector 1120 may have an elbow (e.g., a 90° bend) proximate the outlet 1122 to direct the flow from the tubular portion 1128 through the outlet 1122 and the bandage 1002. The flange 1130 may extend at least partially around the outlet 1122 to attach the first connector 1120 to the bandage 1002.

[0292] The injection port 1100 may be attached to the bandage 1002 with at least one adhesive. For example, the injection port 1100 may be (directly or indirectly) connected to the distal surface 1042 of the second layer 1040 at two or more spaced-apart locations, as illustrated in FIG. 6. The flange 1130 may be attached to the distal surface 1142 of the second layer 1040 with a first adhesive 1070. The first adhesive 1070 may be substantially annular (e.g., a washer) and extend at least partially (e.g., entirely) around the opening 1152 of the second layer 1040. The first adhesive 1070 may be applied against the proximal surface of the flange 1130, and the protrusion 1121 may extend the thickness of the first adhesive 1070 to prevent the first adhesive 1070 from contacting the biomaterial passing through the outlet 1122 to avoid wetting. The first adhesive 1070 may be (directly or indirectly) adhered to the distal surface 1042 of the second layer 1040. For example, the first adhesive 1070 may be adhered to the stop 1062 that is secured to the distal surface 1042 of the second layer 1040

[0293] The tab 1132 may have an elongated shape and extend substantially perpendicular to the tubular portion 1128. The tab 1132 may be (directly or indirectly) attached to the distal surface 1142 of the second layer 1040 with a second adhesive 1072. As further illustrated in FIGS. 12-14, the tab 1132 may extend across the tubular portion 1128 to provide a secure attachment on both sides of the tubular portion 1128. The second adhesive 1072 may have anAttorney Docket No. 005042-00013elongated, tab shape to attach the tab 1132 to the second layer 1040. The first adhesive 1070 and the second adhesive 1072 may be spaced apart along the length of the tubular portion 1128. The two points of attachment may reduce strain on the attachment of the first connector 1120 to the second layer 1040.

[0294] The injection port 1100 may be releasably attached to the bandage 1002 to be removed from the bandage 1002 after injection of the biomaterial. The tab 1132 may have a projection 1134 configured to facilitate removal of the first connector 1120. As illustrated in FIG. 14, the projection 1134 may have an inner surface 1136 distally spaced from the distal surface 1042 of the second layer 1040. The inner surface 1136 may allow a user to lift, pry, or peel the tab 1132 from the distal surface 1042 of the second layer 1040 to remove the first connector 1120 and the injection port 1100 from the bandage 1102. The inner surface 1136 may extend substantially at an acute angle a relative to the distal surface 1042 (e.g., y-axis) of the second layer 1040 to create a space to receive a finger of the user for the lifting, prying, or peeling motion. The acute angle a may be from about 20° to about 60° relative to the y-axis of the second layer 1140. In some embodiments, the acute angle a may be about 45° relative to the y-axis of the second layer 1140. Lifting the inner surface 1136 may be configured to allow removal of the injection port 1100 from the two or more locations due to a rigidity of the first connector 1120.

[0295] The first adhesive 1070 and the second adhesive 1072 may configured to facilitate the removal of the injection port 1100. Each of the first adhesive 1070 and the second adhesive 1072 may have a first side that has a greater tackiness or adhesiveness than a second side for a controlled release of the first connector 1120. For example, each of the first adhesive 1070 and the second adhesive 1072 may have a distal side that attaches to the first connector 1120 and a proximal side that attaches to the second layer 1040. The distal side have a greater tackiness or adhesiveness than the proximal side to ensure that the adhesive 1070, 1072 is removed from the bandage 1102 with the first connector 1120. The first adhesive 1070 and / or the second adhesive 1072 may comprise a double-sided tape that secures the first connector 1120 to the second layer 1040. The first adhesive 1070 and / or the second adhesive 1072 may be a tape formed of a hydrogel and / or silicon gel. The hydrogel and / or silicon gel may be coated onto a carrier comprising a polyurethane and / or polyester film. The first adhesive 1070 and / or the second adhesive 1072 may further have an acrylic layer and a polyester layer. The acrylic layer and the polyester layer may have different adhesive properties. The acrylicAttorney Docket No. 005042-00013layer may have a stronger adherence than the polyester layer. The acrylic layer may be attached to a proximal surface of the injection port 1100, and the polyester layer may be attached to the distal surface 1042 of the second layer 1040.

[0296] The second layer 1040 may have a surface area of a square, circular, oval, rounded square, or rectangular shape. In some embodiments, the first layer 1020 and / or the second layer 1040 may a length, width, and / or maximum diameter of about 0.5 cm to about 100 cm. In some embodiments, the first layer 1020 may have a length of about 5 cm and a width of about 5 cm, and the second layer 1040 may have a length of about 10 cm and a width of about 10 cm. In some embodiments, the first layer 1020 may have a length of about 8 cm and a width of about 2 cm, and the second layer 1040 may have a length of about 10 cm and a width of about 10 cm. In some embodiments, the first layer 1020 may have a length of about 15 cm and a width of about 15 cm, and the second layer 1040 may have a length of about 8 cm and a width of about 2 cm.

[0297] For example, the wound dressing 1000 may have a round shape. In some embodiments, the wound dressing 1000 may have a diameter of about 3 cm. In another example, the wound dressing 1000 may have an oval shape, a length of about 10 cm, a width of about 3 cm. In another example, the wound dressing 1000 configured to treat a linear wound may have a rectangular shape, a length of about 8 cm and a width of about 2 cm. In another example, the wound dressing 1000 configured to treat a large wound may have a square shape, a length of about 6 cm and a width of about 6 cm.

[0298] In some embodiments, the first layer 1020 may have an additive. The additive may be a coagulant, a stabilizing agent, an anticoagulant, a drying agent and / or an antibacterial agent. The additive may be configured to contact the biomaterial. For example, the additive may be a coagulating agent, such as a calcium salt, an aluminosilicate (e.g., kaolin), diatomaceous earth, a polysaccharide, and / or a polysaccharide (e.g., chitosan). The calcium salt may be calcium gluconate and / or calcium chloride. The coagulating agent may be configured to coagulate or solidify the biomaterial. The stabilizing agent may be ascorbic acid, sodium ascorbate, lactate, gluconic acid, and / or citrate. The anticoagulating agent may be buffered citrate, sodium citrate, citrate phosphate dextrose (CPD), ethylenediaminetetraacetic acid (EDTA), heparin, oxalate, anticoagulant citrate dextrose A (ACD-A), any derivatives thereof, or any combinations thereof. The anticoagulant may inhibit the coagulation of the biomaterial (e.g., PRP or PRFM) when injected. The additive may also be present in the form of aAttorney Docket No. 005042-00013coating, a liquid, a powder, a pellet, and / or other particulates. The additive may be coated on the proximal surface 1024 of the first layer 1020. In some embodiments, the additive may be spray-coated onto the proximal surface 1024 of the first layer 1020 in a particulate form. In some embodiments, the additive may be kaolin in an amount of from about 0.5 mg to about 2000 mg.

[0299] The present disclosure also provides a kit including the wound dressing 1000. The kit may include the bandage 1002 and / or the injection port 1100 (attached together or separated), at least one sterilization, at least one needle, and / or a biomaterial dispenser. In some embodiments, the kit may include solutions and containers to form PRP and / or PRFM. For example, the kit may include a centrifuge tube including one or more separator gel (e.g., thixotropic) to separate a blood sample into components. In some embodiments, the centrifuge tube may be pre-filled with drug layers, such as an anticoagulant, a coagulating agent, and / or a stabilizing agent.

[0300] FIG. 15 illustrates a method 500 of applying a biomaterial to a wound.

[0301] In some embodiments, the biomaterial is a blood-derived product such as PRP or PRFM. As further discussed with reference to FIGS. 1 A-1D and 3A-D (as incorporated herein for sake of brevity), the method may initially include producing the PRP or PRFM. For example, the method 500 may include separating PPP from a blood sample through centrifugation with one or more separator gels. Platelets may then be resuspended into the PPP to form PRP. In some embodiments, the method may include activating the plasma and / or platelets to form PRFM in the centrifuge tube. The biomaterial may then be transferred to a container to be applied to the wound. The container may be a syringe, a hand pump, an electric pump, and / or a mechanical pump.

[0302] In step 502, the wound dressing 1000 may be applied to the subject (e.g., over a wound). Prior to adhering the wound dressing to the subject, the method may include sterilizing the application site (e.g., the wound site) of the subject using a sterilization solution or wipe and allowing the application site to dry. The sterilization may provide better adhesion of the wound dressing 1000 on the application site while also reducing potential contamination of the biomaterial. The wound dressing 1000 may then be removed from a packaging.Attorney Docket No. 005042-00013

[0303] As discussed above, the liner 1200 may be removed from a proximal surface of the bandage 1002 to expose the first layer 1020 and the adhesive 1080 on the proximal surface 1044 of the second layer 1040. The bandage 1002 may then be applied over the wound. The first layer 1020 may be positioned to substantially cover the wound. The perimeter portion 1048 of the proximal surface 1044 may be positioned around the wound, and the adhesive 1080 on the perimeter portion 1048 may secure the bandage 1002 on the subject.

[0304] In step 504, the container containing the biomaterial may be attached to the injection port 1100. The container may have a chamber containing the biomaterial. The container may be configured to releasably attach to the inlet port 1162 of the second connector 1160. In some embodiments, the container may have a Luer connector configured to be attached to a luer connector on the inlet port 1162.

[0305] In step 506, the biomaterial may be injected from the container through the injection port 1100 and the bandage 1002. The biomaterial may pass through the second connector 1160, the tube 1140, and the first connector 1120. In some embodiments, the tube 1140 may be substantially rigid providing a linear passage of the biomaterial to reduce resistance of the biomaterial. In some embodiments, the tube 1140 may be flexible to facilitate attachment of the container. The first connector 1120 may guide the biomaterial with the bend proximally toward the wound. The biomaterial may push the valve 1060 open to enable passage through the second opening 1052 of the second layer 1040 and the first opening 1032 of the first layer 1020. The biomaterial may displace gas (e.g., air) that passes through the first pores 1030 and the second pores 1050 as the biomaterial is passed through bandage 1002 and into contact with the wound. The gas may be vented through the first pores 1030 and the second pores 1050. The valve 1060 may close when the biomaterial is completely passed the valve 1060 to retain the biomaterial against the wound. The biomaterial may be in a liquid state, a gel-like, gel, and / or solid state when delivered. The injection port 1100 and openings 1032, 1052 may be sized for the passage of biomaterial with various viscosities. The wound dressing 1000 may be particularly suitable for viscous, liquid biomaterials due to the larger size of the first opening 1032 and the second opening 1052, and the first pores 1030 retaining the biomaterial against the wound. For example, the biomaterial, when injected, may have a viscosity of up to 100 cP.

[0306] In step 508, the injection port 1100 may be removed from the bandage 1002. For example, the user may peel the first connector 1120 from the second layer 1040 by lifting theAttorney Docket No. 005042-00013projection 1134 of the release tab 1132. The first adhesive 1170 and the second adhesive 1172 may detach from the second layer 1040 to be removed with the injection port 1100. The bandage 1102 may remain in place to retain the biomaterial against the wound and to cover and protect the wound. Removal of the injection port 1100 may ensure comfort of the subject as the bandage 1102 covers the wound and the wound heals.

[0307] In step 510, the biomaterial may solidify and / or coagulate and be vented through the bandage 1102. The ventilation of the bandage 1102 may facilitate the solidification and / or coagulation by providing a sufficient airflow. The time required for coagulation of the biomaterial may be about 10 minutes to about 2 hours. The biomaterial may conform to the wound as it solidifies and / or coagulates. The biomaterial (e.g., PRP and / or PRFM) may provide a fibrous scaffold rich in structural protein and growth factors improving the healing of the wound.

[0308] In step 512, the second layer 1040 may be removed from the first layer 1020. The second layer 1040 may be removed before or after the biomaterial solidifies and / or coagulates. For example, the second layer 1040 may provide ventilation to the biomaterial as it solidifies and / or coagulates, and then the second layer 1040 may be removed. The second layer 1040 may be removed about 12 hours to about 4 days after the biomaterial is applied to the wound. The second layer 1040 may be removed by peeling the second layer 1040 off of the skin and the first layer 1020, detaching the adhesive 1080 from each. The first layer 1020 may be retained after the second layer 1040 is removed to protect the wound and / or retain the biomaterial. An additional wrap or bandage may retain the first layer 1020 in place. The additional wrap or bandage may be adhesive free to prevent irritation of the skin.

[0309] In step 514, the first layer 1020 may be removed from the wound. The first layer 1020 may be removed from the subject after the wound is substantially healed. For example, the first layer 1020 may be removed about 4 days to about 14 days after the biomaterial is applied to the wound.

[0310] FIGS. 16-18 illustrate a second embodiment of a wound dressing 1000’. The wound dressing 1000’ may have one or more of the same features as disclosed for the wound dressing 1000, the entire disclosure of which is incorporated herein for sake of brevity. For example, the wound dressing 1000’ may have all of the features of the wound dressing 1000 as disclosed. The wound dressing 1000’ may be used in the method 500, as discussed.Attorney Docket No. 005042-00013

[0311] As further illustrated, the wound dressing 1000’ may further include at least one nonadhesive insert 1300 configured to facilitate separation of the second layer 1040 from the first layer 1020 to allow removal of the second layer 1040 while maintaining the first layer 1020 at the wound site, for example as discussed with reference to Step 512. The non-adhesive insert 1300 may provide an area of non-adhesion to facilitate fingers grabbing a corner of the first layer 1020. The non-adhesive insert 1300 may be positioned between the proximal surface 1044 of the second layer 1040 and the distal surface 1022 of the first layer 1020 to ensure that the adhesive 1080 of the second layer 1040 does not stick to a portion of the first layer 1020. For example, as illustrated, the non-adhesive insert 1300 may positioned at a comer of the first layer 1020 to facilitate peeling of the second layer 1040 at the corner of the wound dressing 1000’. When in position, the non-adhesive insert 1300 may extend laterally from the first layer 1020 outside of the perimeter of the first layer 1020 to allow the user to wedge a finger between the non-adhesive insert 1300 and the second layer 1040. As illustrated, the non-adhesive insert 1300 may be positioned at a single corner of the first layer 1020 to provide an asymmetric peeling advantage of the second layer 1040. For example, the comer of the first layer 1020 may be indicated by the corresponding corner of the second layer 1040. The corner of the second layer 1040 may have a peel tab (not shown) indicating alignment with the corner of the first layer 1020 having the non-adhesive insert 1300.Additionally or alternatively, the corner of the second layer 1040 may be indicated by another indicator, such as a visual indicator (e.g., a marking and / or indentation) and / or tactile indicator (e.g., a textured pattern) on the distal surface 1042 of the second layer 1040.Additionally or alternatively, the wound dressing 1000’ may include a plurality of the non-adhesive inserts 1300. For example, when the first layer 1020 and / or the second layer 1040 is a quadrilateral (as shown), a plurality of the comers (e.g., each corner) of the first layer 1020 may each have a non-adhesive insert 1300.

[0312] FIGS. 16 and 17 illustrate a third embodiment of a wound dressing 3000. The wound dressing 3000 may have one or more of the same features as the previously disclosed embodiments of the wound dressing 1000, 1000’, the entire disclosures of which are incorporated herein for sake of brevity. The wound dressing 3000 may be used in the method 500, as discussed.

[0313] As illustrated, the wound dressing 3000 may include a housing 3100 and a cover 3300.Attorney Docket No. 005042-00013

[0314] The housing 3100 may have a frame 3120 and a ceiling 3140. In some embodiments, the frame 3120 and the ceiling 3140 may be made of one-piece. In some embodiments, the frame 3120 and the ceiling 3140 may be made of two pieces fixed together. The ceiling 3140 may be a distal portion of the frame 3120. The ceiling 3140 may have a proximal surface that is configured to face the skin of the subject when the wound dressing 3000 is applied. In some embodiments, the ceiling 3140 defines a flat or contoured surface. A distal surface of the ceiling 3140 may configured to face towards the environment and away from the skin. The ceiling 3140 may include a plurality of ribs, a raised ceiling (e.g., an outward projecting dome), and / or a surface finish (e.g., pits, bumps, and / or groves (e.g. to resemble skin texture)).

[0315] The frame 3120 may extend vertically from the skin to separate the ceiling 3140 from the skin and form a cavity 3160 between the ceiling 3140 and the skin. The cavity 3160 may have a width, length, and / or a maximum diameter of about 0.5 cm to about 50 cm. The length and width of the cavity 3160 may be the same or different. The cavity 3160 may have a circular, oval, rounded square, or rectangular shape. The dimensions and / or shape of the cavity 3160 may be defined by the size, depth, volume, and / or shape of the wound. The cavity 3160 may serve as a mold for the biomaterial as it dries and / or coagulates, such that the biomaterial may be formed in the shape of the cavity 3160 upon removal of the wound dressing 3000. The biomaterial may be molded by the shape of a plurality of ribs, a raised ceiling, and / or a surface finish. The plurality of ribs may be hidden behind a reveal. The plurality of ribs may be configured to allow an expansion of the volume of the cavity 3160. For example, once the volume of biomaterial injected into the cavity 3160 has reached the minimum fill volume of the cavity 3160, the biomaterial may flow into the plurality of ribs, thereby expanding the cavity 3160. The additional volume of biomaterial may be injected to form a textured distal surface of the biomaterial, such that the texture is formed by the plurality of ribs and / or the surface finish. The raised ceiling may also allow an additional volume of biomaterial beyond that of the minimum fill volume to be injected into the cavity 3160. The biomaterial may then conform to the shape of the frame 3120 and / or the ceiling 3140. For example, the distal side of the biomaterial may form a dome shape. In some embodiments, the frame 3120 may be omitted such that any vertical frame is omitted and the ceiling 3140 is a flat sheet that lies substantially against the skin of the subject.Attorney Docket No. 005042-00013

[0316] The wound dressing 3000 may have an injection port 3200. The injection port 3200 may have an inlet 3220 and a tube 3240. The injection port 3200 may be configured to introduce the biomaterial into the cavity 3160 through the inlet 3220 and the tube 3240. A valve 3260 configured prevent backflow of the biomaterial. The valve 3260 may be attached to the housing 3100, the inlet 3220, and / or the tube 3240. In some embodiments, the valve 3260 may be attached to the housing 3100, and the injection port 3200 may be removable after the biomaterial is introduced into the housing 3100. The injection port 3200 may have one or of the features as discussed above with respect to the wound dressing 1000, the entire disclosure of which is incorporated herein by reference.

[0317] The housing 3100 may have a plurality of first pores 3150 through the ceiling 3140, as illustrated. The first pores 3150 may be sized to allow for ventilation of the cavity 3160, such as allowing the passage of gas (e.g., air) as the biomaterial is introduced into the housing 3100. In some embodiments, the first pores 3150 may be sized to allow a fluid (e.g., an exudate) to escape from the cavity 3160 while the wound dressing 3000 is in place on the skin after removal of the cover 3300. In some embodiments, the first pores 3150 may further be sized to allow for the biomaterial to be pass therethrough, for example when excess biomaterial was injected into the cavity 3160.

[0318] The cover 3300 may be applied to the distal surface of the housing 3100. The cover 3300 may at least partially cover the first pores 3150. The cover 3300 may be releasably attached to the distal surface of the housing 3100. The cover 3300 may have a plurality of second pores (not shown). In some embodiments, the first pores 3150 may be larger than the second pores of the cover 3300. The second pores may be sized for venting gas (e.g., air) from the cavity 3160 while inhibiting the escape of biomaterial out from the cavity 3160. In some embodiments, the second pores may also be sized for the passage of a fluid (e.g., exudate) from the internal cavity. Alternatively, the second pores in the cover 3300 may be larger than the first pores 3150, as discussed above with respect to the wound dressing 1000, the entire disclosure of which is incorporated herein by reference. The second pores may be distributed across the cover 3300 in distinct central, intermediate, and peripheral zones (e.g., the central zone that overlies the first layer and internal cavity may exhibit the highest coverage of second pores). The second pores may comprise pores that may occupy various density ranges across the entirety of the cover 3300 (e.g., the central region of the cover 3300 may have more pores than the intermediate region or the peripheral region).Attorney Docket No. 005042-00013

[0319] The cover 3300 may be releasably attached to the housing 3100, e.g., with an adhesive. The cover 3300 may be removed (e.g., peeled off) after the biomaterial is injected into the cavity 3160 to a desired volume. The cover 3300 may include a pull-tab extending outside the area of the housing 3100 and / or wound dressing 3000 to allow the cover 3300 to be peeled off. The cover 3300 may be removed once the biomaterial is coagulated or solidified after injection into cavity 3160 (e.g., a few hours (e.g., less than 24 hours) after injection of the biomaterial). The removal of the cover 3300 may expose the pores 3150.

[0320] The cover 3300 may be a plastic sheet or plastic film. The cover 3300 may be made of a polyvinyl chloride (PVC), polyethylene (PE), thermoplastic polyurethane (TPU), or any biocompatible polymer. The cover 330 may include a slit to accommodate (e.g., fit around) the injection port 3200 of the wound dressing.

[0321] The housing 3100 may include an additive 3180 on one or more of the internal walls of the cavity 3160. As discussed above, the additive may be a coagulant, a stabilizing agent, an anticoagulant, a drying agent and / or an antibacterial agent. The additive may be configured to contact the biomaterial. For example, the additive may be a coagulating agent, such as a calcium salt, an aluminosilicate (e.g., kaolin), diatomaceous earth, a polysaccharide, and / or a polysaccharide (e.g., chitosan). The calcium salt may be calcium gluconate and / or calcium chloride. The coagulating agent may be configured to coagulate or solidify the biomaterial. The stabilizing agent may be ascorbic acid, sodium ascorbate, lactate, gluconic acid, and / or citrate. The anticoagulating agent may be buffered citrate, sodium citrate, citrate phosphate dextrose (CPD), ethylenediaminetetraacetic acid (EDTA), heparin, oxalate, anticoagulant citrate dextrose A (ACD-A), any derivatives thereof, or any combinations thereof. The anticoagulating agent inhibits the coagulation of the biomaterial (e.g., PRP or PRFM) in the internal cavity. The additive may also be present in the form of a coating, a liquid, a powder, a pellet, and / or other particulates In some embodiments, the additive may be spray-coated in a particulate form onto the one or more of the internal walls of the cavity 3160.

[0322] At least a portion of the housing 3100 may be optically transparent to visualize the biomaterial as it enters the cavity 3160. Further, the housing 3100 may be flexible, deformable, and / or conformable. The ceiling 3140 may be provided in the form of a sheet, membrane, hydrogel, or a solid material. The housing 3100 may be made from a polymer, a silicon, a foam, a mesh, a hydrogel, any derivatives thereof, or any combinations thereof. TheAttorney Docket No. 005042-00013polymer and / or hydrogel may be made from or may include polyethylene, polyester, vinyl, and / or any biocompatible materials (e.g., silicone or PVC).

[0323] The housing 3100 may have a proximal contact surface coated with an adhesive 3020. The adhesive 3020 may seal the cavity 3160 from the outside environment when applied to skin. The adhesive 3020 may comprise a low-tack adhesive to enable removal of the wound dressing 3000 from the subject. The adhesive 3020 may be a silicon gel adhesive.

[0324] A liner 3010 may be configured to cover the adhesive 3020 and enclose the cavity 3160. The liner 3010 may be removed prior to use to expose the adhesive 3020 and the cavity 3160. In some embodiments, the liner 3010 may be a single sheet. In some embodiments, the liner 3010 may be a split liner, as discussed with reference to the wound dressing 1000, the entire disclosure of which is incorporated herein.

[0325] FIGS. 21-23 illustrate a fourth embodiment of a wound dressing 4000. The wound dressing 4000 may have one or more of the features as the previously disclosed embodiments of the wound dressing 1000, 1000’, and / or 3000, the entire disclosure of which is incorporated herein for sake of brevity. For example, the wound dressing 4000 may include one or more features of the wound dressing 3000. The wound dressing 4000 may be used in the method 500, as discussed. As further illustrated, the wound dressing 4000 may include a housing 4100 having a frame 4120 and a ceiling 4140. The housing 4100 may further include a skirt 4102 extending at least partially around the perimeter of the housing 4100 for increased surface contact between the wound dressing and the application site. An injection port 4200 may extend through a distal surface of the ceiling 4140. For example, the injection port 4200 may extend substantially perpendicular to a surface area of the ceiling 4140.

[0326] FIG. 24 illustrates a fifth embodiment of a wound dressing 4000’. The wound dressing 4000’ may have one or more of the same features as the previously disclosed embodiments of the wound dressing 1000, 1000’, 3000, and / or 4000, the entire disclosures of which is incorporated herein for sake of brevity. For example, the wound dressing 4000’ may have all of the features of the wound dressing 3000 and / or the wound dressing 4000, as disclosed. The wound dressing 4000’ may be used in the method 500, as discussed. As further illustrated, the wound dressing 4000’ may include a housing 4100 having a frame 4120 and a ceiling 4140. An injection port 4200 may extend through a distal surface of the ceiling 4140. For example, the injection port 4200 may extend substantially perpendicular to a surface areaAttorney Docket No. 005042-00013of the ceiling 4140. The wound dressing 4000’ may further include one or more pull tabs 4002’ to remove the wound dressing 4000’ from the subject.

[0327] FIGS. 25 and 26 illustrate a sixth embodiment of a wound dressing 6000. The wound dressing 6000 may have one or more of the same features as the previously disclosed embodiments of the wound dressing 1000, 1000’, 3000, 4000, and / or 4000’, the entire disclosures of which is incorporated herein for sake of brevity. For example, the wound dressing 6000 may include features of the wound dressing 1000 (as discussed with reference to FIGS. 4-14) and / or the wound dressing 3000 (as discussed with reference to FIGS. 19-20). The wound dressing 6000 may be used in the method 500, as discussed.

[0328] As further illustrated, the wound dressing 6000 may have a first inlet port 6200 and a second inlet port 6250, each in communication with a chamber and / or area proximal of the wound dressing 6000. The first inlet port 6200 may be configured to be coupled to a first syringe 50, and the second inlet port 6250 may be configured to be coupled to a second syringe 52. For example, the first syringe 50 may be configured to deliver the biomaterial through the wound dressing 600. The second syringe 52 may be configured to pull fluid and / or air through the wound dressing 600, for example as the biomaterial is injected.

[0329] FIGS. 27-29 illustrate a seventh embodiment of a wound dressing 7000. The wound dressing 7000 may have one or more of the same features as the previously disclosed embodiments of the wound dressing 1000, 1000’, 3000, 4000, 4000’, and / or 6000, the entire disclosures of which is incorporated herein for sake of brevity. The wound dressing 7000 may be used in the method 500, as discussed.

[0330] The wound dressing 7000 may include a housing 7100, a cover 7300, and an insert 7500. The housing 7100 may include a plurality of first pores 7150 and a cavity (not shown), for example as discussed with reference to the wound dressing 300 of FIGS. 19 and 20, the entirely disclosure of which is incorporated herein for sake of brevity. The insert 7500 may be positioned between the housing 7100 and the cover 7300. The insert 7500 may be releasably coupled to the housing 7100. The insert 7500 may include an injection port 7520 configured to be in communication with an opening 7152 of the housing 7100 to provide access to the cavity of the housing 7100. The cover 7300 may include a slit 7302 configured to accommodate the injection port 7520 for access to the injection port 7520 with the cover 7300 placed over the housing 7100.Attorney Docket No. 005042-00013

[0331] As illustrated in FIGS. 27, the injection port 7520 may be accessed through the cover 7300 for injection of the biomaterial with a container (e.g., a syringe). For example, the injection port 7520 may include a Luer lock or Luer slip configured to connect to a container (e.g., a syringe). Before, during and / or after the biomaterial is injected into the opening 7152, the cover 7300 may be lifted from the housing 7100. For example, the cover 7300 may be attached to the housing 7100 on one side to provide a pivotable connection between the cover 7300 and the housing 7100. In some embodiments, the cover 7300 may be kept open for a period of time to expose the first pores 7150 to ventilate the cavity (e.g., to promote the transition of biomaterial within cavity from a liquid state to a gel state), as discussed herein. The insert 7500 may have one or more pores and / or a complete cutout exposing the first pores of the housing 7100 to the environment. A valve (not shown) may be configured prevent backflow of the biomaterial. The valve may be attached to the housing 7100, the injection port 7520 and / or the insert 7500. In some embodiments, the valve may be attached to the housing 7100 and be configured to close the opening 7152 after removal of the insert 7500, as discussed above with respect to the wound dressing 1000 (the entire disclosure of which is incorporated herein by reference). In some embodiments, the cover 7500 may be placed back over the housing 7100, for example to close the pores 7150 and / or the opening 7152.

[0332] FIGS. 30 and 31 illustrate a sixth embodiment of a wound dressing 8000. The wound dressing 6000 may have one or more of the same features as the previously disclosed embodiments of the wound dressing 1000, 1000’, 3000, 4000, 4000’, 6000, and / or 7000 the entire disclosure of which is incorporated herein for sake of brevity. The wound dressing 8000 may be used in the method 500, as discussed.

[0333] As further illustrated, the wound dressing 8000 may have a bandage 8002 and an injection port 8200. The bandage 8002 and / or the injection port 8200 may have one or more features of the injection port of the other embodiments as disclosed herein, the entire disclosure of which is incorporated herein. The injection port 8200 may have an inlet 8204 configured to be coupled to a container of biomaterial to receive the biomaterial. The injection port 8200 may further have a plurality of pores 8202 sized to vent gas (e.g., air). The inlet assembly 8200 may allow ventilation of the gas through the pores 8202 during and / or after the biomaterial is introduced into and / or through the wound dressing, as discussed herein. The pores 8202 may replace or supplement pores of the bandage 8002. TheAttorney Docket No. 005042-00013ventilation features of the injection port 8200 may be implemented in any of the previously disclosed embodiments.

[0334] Kits are disclosed herein including one or more of the pre-filled tubes 100, 100’ and one or more of the wound dressings 1000, 1000’, 3000, 4000, 4000’, 6000, 7000, and / or 8000, as discussed herein. For example, the pre-filled tube 100 and the wound dressing 1000 or 1000’ may be included in a kit to be used to treat a wound. The various embodiments of the kits may include one or more containers (e.g., syringes) for transferring the fluid (e.g., blood) products. The various embodiments of the kits may further include a needle (e.g., a butterfly needle) and / or antiseptic wipes to withdrawn the blood sample from the patient. The various embodiments of the kits may include additional components, as disclosed herein. The kits may be packaged together and / or separately.OTHER EMBODIMENTS

[0335] Other embodiments are in the claims. All publications, patents, and patent applications mentioned in the above specification are hereby incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.

[0336] Other embodiments are within the claims.

Claims

Attorney Docket No. 005042-00013CLAIMS1. A method compri sing :introducing a blood sample into a tube, wherein the blood sample, when in the tube, is separated from a coagulating agent with at least one gel layer;centrifuging the tube to pass red blood cells of the blood sample through the at least one gel layer and to form a mixture of the coagulating agent and plasma of the blood sample positioned above the at least one gel layer;resuspending platelets of the blood sample into the mixture to form a platelet rich fibrin matrix (PRFM) layer in the tube;removing at least a portion of the PRFM layer from the tube in a fluid state with a syringe;applying a wound dressing at least partially over a wound; andinjecting the at least the portion of the PRFM layer through an opening of the wound dressing, wherein air is vented through pores of the wound dressing.

2. The method of claim 1, wherein the at least the portion of the PRFM layer is removed from the tube having a viscosity less than about 100 cP.

3. The method of claim 1, further comprising mixing a stabilizing agent with the coagulating agent and the plasma in the tube.

4. The method of claim 1, further comprising mixing the blood sample with an anticoagulant in the tube before centrifuging.

5. The method of claim 1, wherein the centrifuging is the only centrifugation.

6. The method of claim 1, wherein the injecting is through a one-way valve of the wound dressing.

7. The method of claim 1, wherein air is vented through a plurality of first pores of a first layer of the wound dressing and one or more second pores of a second layer of the wound dressing.

8. The method of claim 1, wherein the injecting is through an injection port attached to a distal surface of the wound dressing.Attorney Docket No. 005042-000139. The method of claim 8, further comprising removing the injection port from the distal surface of the wound dressing after injecting.

10. The method of claim 9, wherein the removing the injection port comprises lifting a tab of the injection port.

11. A kit compri sing :a pre-filled tube configured to prepare a platelet rich fibrin matrix (PRFM) layer, wherein the pre-filled tube comprises:a first drug layer comprising a coagulating agent;a second drug layer comprising a stabilizing agent; anda gel layer separating the first drug layer and the second drug layer; anda wound dressing for applying a biomaterial to a subject, the wound dressing comprising:a first layer comprising a first opening and a plurality of first pores having a first width; anda second layer comprising a second opening and one or more second pores having a second width,wherein the first layer is configured to be between the second layer and the subject when the wound dressing is applied to the subject, the first opening is at least partially aligned with the second opening to allow passage of the biomaterial through the first layer and the second layer, and the second width is larger than the first width.

12. A method compri sing :introducing a blood sample into a tube, wherein the blood sample, when in the tube, is separated from a stabilizing agent and a coagulating agent with at least one gel layer;centrifuging the tube to pass red blood cells of the blood sample through the at least one gel layer and to form a mixture of the stabilizing agent, the coagulating agent, and plasma of the blood sample positioned above the at least one gel layer; andresuspending platelets of the blood sample into the mixture to form a platelet rich fibrin matrix (PRFM) layer in the tube.

13. The method of claim 12, further comprising:removing at least a portion of the PRFM layer from the tube in a fluid state with a syringe; andAttorney Docket No. 005042-00013applying the at least the portion of the PRFM layer to a wound site.

14. The method of claim 13, wherein the PRFM layer is removed from the tube having a viscosity less than about 100 cP.

15. The method of claim 12, further comprising:removing at least a portion of the PRFM layer from the tube in a gel state; and applying the at least the portion of the PRFM layer to a wound site.

16. The method of claim 15, wherein the PRFM layer is removed from the tube having a viscosity greater than about 100 cP.

17. The method of claim 12, wherein the coagulating agent activates the platelets to form the PRFM layer, and the stabilizing agent stabilizes the PRFM layer.

18. The method of claim 12, wherein the at least one gel layer includes a first gel layer separating the coagulating agent from the stabilizing agent and a second gel layer separating the coagulating agent and the stabilizing agent from the blood sample.

19. The method of claim 12, further comprising mixing the blood sample with an anticoagulant in the tube before centrifuging.

20. The method of claim 19, wherein the anticoagulant comprises anticoagulant citrate dextrose A (ACD-A), buffered citrate, sodium citrate, citrate phosphate dextrose (CPD), ethylenediaminetetraacetic acid (EDTA), heparin, oxolate, a derivative thereof, or any combination thereof.

21. The method of claim 20, wherein the anticoagulant comprises ACD-A.

22. The method of claim 12, wherein the stabilizing agent comprises ascorbic acid.

23. The method of claim 12, wherein the coagulating agent comprises a calcium salt.

24. The method of claim 23, wherein the calcium salt comprises calcium gluconate and / or calcium chloride.

25. The method of claim 12, wherein the at least one gel layer comprises at least one thixotropic gel.Attorney Docket No. 005042-0001326. The method of claim 12, wherein the tube is prefilled with the stabilizing agent, the coagulating agent, and the at least one gel layer.

27. The method of claim 26, wherein the tube is prefilled with an anti -coagulant agent.

28. The method of claim 26, wherein the tube contains a vacuum and is closed by a sealing cap.

29. The method of claim 26, wherein the stabilizing agent is prefilled in the tube at a concentration of from about 400 mg / mL to about 600 mg / mL, inclusive.

30. The method of claim 26, wherein the coagulating agent is prefilled in the tube at a concentration of from about 50 mg / mL to about 150 mg / mL, inclusive.

31. The method of claim 12, wherein the centrifuging is at an acceleration of from about 1000 g to about 4500 g, inclusive.

32. The method of claim 12, wherein the centrifuging is for a time of from about 3 minutes to about 12 minutes, inclusive.

33. The method of claim 12, wherein the centrifuging is the only centrifugation.

34. A pre-filled tube configured to prepare a platelet rich fibrin matrix (PRFM) layer, wherein the pre-filled tube comprises:a first drug layer comprising a coagulating agent;a second drug layer comprising a stabilizing agent; anda gel layer separating the first drug layer and the second drug layer.

35. The pre-filled tube of claim 34, wherein the pre-filled tube further comprises:a third drug layer comprising an anticoagulant; anda second gel layer separating the third drug layer from the first drug layer and the second drug layer.

36. The pre-filled tube of claim 35, wherein the anticoagulant comprises anticoagulant citrate dextrose A (ACD-A), buffered citrate, sodium citrate, citrate phosphate dextrose (CPD), ethylenediaminetetraacetic acid (EDTA), heparin, oxolate, a derivative thereof, or any combination thereof.Attorney Docket No. 005042-0001337. The pre-filled tube of claim 36, wherein the anticoagulant comprises ACD-A.

38. The pre-filled tube of claim 34, wherein the coagulating agent comprises a calcium salt.

39. The pre-filled tube of claim 38, wherein the calcium salt is calcium gluconate and / or calcium chloride.

40. The pre-filled tube of claim 34, wherein the coagulating agent has a concentration of from about 50 mg / mL to about 150 mg / mL, inclusive.

41. The pre-filled tube of claim 34, wherein the stabilizing agent is ascorbic acid.

42. The pre-filled tube of claim 34, wherein the stabilizing agent has a concentration of from about 400 mg / mL to about 600 mg / mL, inclusive.

43. The pre-filled tube of claim 34, wherein the first drug layer is closer to a distal end of the pre-filled tube than the second drug layer.

44. The pre-filled tube of claim 34, wherein the second drug layer is closer to a distal end of the pre-filled tube than the first drug layer.

45. The pre-filled tube of claim 34, wherein the pre-filled tube is sealed at a proximal end with a sealing cap to create a vacuum inside of the pre-filled tube.

46. The pre-filled tube of claim 45, wherein the pre-filled tube contains an air pressure from about negative 50 kPa to about negative 101 kPa, inclusive.

47. A pre-filled tube configured to prepare a platelet rich fibrin matrix (PRFM) layer, wherein the pre-filled tube contains five layers of liquid, gel, or solid material and no more than five layers of liquid, gel, or solid material, the five layers of liquid, gel, or solid material comprising:a first drug layer comprising a coagulating agent;a second drug layer comprising a stabilizing agent;a third drug layer comprising an anticoagulant;a first gel layer separating the first drug layer and the second drug layer; and a second gel layer separating the third drug layer from the first drug layer and the second drug layer,Attorney Docket No. 005042-00013wherein the pre-filled tube is sealed at a proximal end with a sealing cap to create a vacuum inside of the pre-filled tube.

48. The pre-filled tube of claim 47, wherein the first drug layer is closer to a distal end of the pre-filled tube than the second drug layer.

49. The pre-filled tube of claim 47, wherein the second drug layer is closer to a distal of the pre-filled tube than the first drug layer.

50. The pre-filled tube of claim 47, wherein the anticoagulant comprises anticoagulant citrate dextrose A (ACD-A), buffered citrate, sodium citrate, citrate phosphate dextrose (CPD), ethylenediaminetetraacetic acid (EDTA), heparin, oxolate, a derivative thereof, or any combination thereof.

51. The pre-filled tube of claim 50, wherein the anticoagulant comprises ACD-A.

52. The pre-filled tube of claim 47, wherein the coagulating agent comprises a calcium salt.

53. The pre-filled tube of claim 52, wherein the calcium salt is calcium gluconate and / or calcium chloride.

54. The pre-filled tube of claim 53, wherein the calcium salt has a concentration of from about 50 mg / mL to about 150 mg / mL, inclusive.

55. The pre-filled tube of claim 47, wherein the stabilizing agent is ascorbic acid.

56. The pre-filled tube of claim 55, wherein the stabilizing agent has a concentration of from about 400 mg / mL to about 600 mg / mL, inclusive.

57. A pre-filled tube configured to prepare a platelet rich fibrin matrix (PRFM) layer, wherein the pre-filled tube contains five layers of liquid, gel, or solid material and no more than five layers of liquid, gel, or solid material, the five layers of liquid, gel, or solid material comprising:a first drug layer comprising calcium gluconate and / or calcium chloride in a concentration of about 50 mg / mL to about 150 mg / mL;a second drug layer comprising ascorbic acid in a concentration of from about 400 mg / mL to about 600 mg / mL;Attorney Docket No. 005042-00013a third drug layer comprising an anticoagulant;a first gel layer separating the first drug layer and the second drug layer; and a second gel layer separating the third drug layer from the first drug layer and the second drug layer,wherein the pre-filled tube is sealed at a proximal end with a sealing cap to create a vacuum inside of the pre-filled tube.

58. A pre-filled tube configured to prepare a platelet rich fibrin matrix (PRFM) layer, wherein the pre-filled tube comprises:a first drug layer comprising a coagulating agent;a second drug layer comprising a stabilizing agent;a third drug layer comprising an anticoagulant;a first gel layer; anda second gel layer,wherein one of the first drug layer and the second drug layer has a distal end that contacts the pre-filled tube and a proximal end that contacts the first gel layer, another of the first drug layer and the second drug layer has a distal end that contacts the first gel layer and a proximal end that contacts the second gel layer, and the third drug layer has a distal end that contacts the second gel layer and a proximal end that contacts a space in the pre-filled tube.

59. The pre-filled tube of claim 58, wherein the pre-filled tube is sealed at a proximal end with a sealing cap to create a vacuum inside of the pre-filled tube.

60. A wound dressing for applying a biomaterial to a subject, the wound dressing comprising:a first layer comprising a first opening and a plurality of first pores having a first width; anda second layer comprising a second opening and one or more second pores having a second width,wherein the first layer is configured to be between the second layer and the subject when the wound dressing is applied to the subject, the first opening is at least partially aligned with the second opening to allow passage of the biomaterial through the first layer and the second layer, and the second width is larger than the first width.Attorney Docket No. 005042-0001361. The wound dressing of claim 60, wherein at least a subset of the first pores is at least partially aligned with the one or more second pores to allow for ventilation through the first layer and the second layer.

62. The wound dressing of claim 60, wherein a distal surface of the second layer is at least partially uncovered to allow for ventilation through the second layer directly to the atmosphere.

63. The wound dressing of claim 62, wherein the one or more second pores are uncovered distally and directly open to the atmosphere.

64. The wound dressing of claim 60, wherein the first width is from about 0.2 microns to about 500 microns, inclusive.

65. The wound dressing of claim 60, wherein the second width is from about 1 mm to about 10 mm, inclusive.

66. The wound dressing of claim 60, wherein the first layer comprises a mesh.

67. The wound dressing of claim 66, wherein the mesh is non-absorbent.

68. The wound dressing of claim 60, further comprising a valve configured for one-way passage of the biomaterial through the first opening and / or the second opening.

69. The wound dressing of claim 68, wherein the valve is attached to the second layer.

70. The wound dressing of claim 69, wherein the valve is a flap integral to the second layer and formed by a slit.

71. The wound dressing of claim 68, further comprising a stop configured to limit distal movement of the valve.

72. The wound dressing of claim 60, wherein the second layer has a first perimeter larger than a second perimeter of the second layer.

73. The wound dressing of claim 60, further comprising an adhesive on a proximal surface of the second layer.Attorney Docket No. 005042-0001374. The wound dressing of claim 73, wherein the adhesive adheres the first layer to the second layer.

75. The wound dressing of claim 73, wherein the adhesive is on a peripheral portion of the second layer to adhere the second layer to the subject.

76. The wound dressing of claim 73, wherein the adhesive comprises silicon.

77. The wound dressing of claim 73, further comprising a non-adhesive insert positioned between the adhesive and the first layer, wherein the non-adhesive insert is configured to facilitate release of the second layer from the first layer.

78. The wound dressing of claim 60, further comprising an injection port attached to a distal surface of the second layer, wherein the injection port is configured to introduce the biomaterial through the first opening and the second opening.

79. The wound dressing of claim 78, wherein the injection port is attached to the distal surface of the second layer with at least one adhesive.

80. The wound dressing of claim 79, wherein the at least one adhesive includes a first adhesive and a second adhesive, the first adhesive is at least partially around the second opening, and the first adhesive is spaced apart from the second adhesive.

81. The wound dressing of claim 80, wherein the injection port has a tab, and the second adhesive adheres the tab to the distal surface of the second layer.

82. The wound dressing of claim 81, wherein the tab is configured to allow a user to peel the injection port from the distal surface of the second layer.

83. The wound dressing of claim 82, wherein the tab comprises a projection having an inner surface spaced apart from the distal surface of the second layer to allow the user to lift the tab from the distal surface of the second layer.

84. The wound dressing of claim 83, wherein the inner surface of the projection is disposed at an acute angle with respect to the distal surface of the first layer.

85. The wound dressing of claim 80, wherein the at least one adhesive is configured to releasably attach the injection port to the distal surface of the second layer.Attorney Docket No. 005042-0001386. The wound dressing of claim 85, wherein the at least one adhesive has a distal surface configured to attach to the injection port and a proximal surface configured to attach to the distal surface of the second layer, and the distal surface of the at least one adhesive has a greater tackiness than the proximal surface of the at least one adhesive to allow for release of the at least one adhesive from the distal surface of the second layer with the injection port.

87. A kit comprising:the wound dressing of claim 60; anda container configured to form or inject the biomaterial.

88. The kit of claim 87, wherein the biomaterial comprises a platelet rich plasma (PRP) or a platelet rich fibrin matrix (PRFM).

89. The kit of claim 87, wherein the container comprises a syringe configured to inject the biomaterial.

90. The kit of claim 87, wherein the container is a centrifuge tube including at least one separator gel configured to form the biomaterial.

91. A wound dressing for applying a biomaterial to a subj ect, the wound dressing comprising:a first layer comprising a first opening and a plurality of first pores having a first width;a second layer comprising a second opening, one or more second pores having a second width, and a flap configured to pivot relative to the second opening; andan injection port releasably attached to a distal surface of the second layer with a first adhesive and a second adhesive, wherein the first adhesive and the second adhesive are spaced apart, and the injection port is configured to introduce the biomaterial through the first opening and the second opening,wherein the first layer is configured to be between the second layer and the subject when the wound dressing is applied to the subject, the first opening is at least partially aligned with the second opening, the flap provides one-way passage of the biomaterial through the second opening, the second width is larger than the first width, and the one or more second pores are uncovered distally and directly open to the atmosphere.Attorney Docket No. 005042-0001392. The wound dressing of claim 91, wherein each of the first adhesive and the second adhesive has a distal surface configured to attach to the injection port and a proximal surface configured to attach to the distal surface of the second layer, and the distal surface has a greater tackiness than the proximal surface to allow for release from the distal surface of the second layer with the injection port.

93. The wound dressing of claim 91, wherein the first adhesive is positioned on the distal surface of the second layer at least partially around the second opening.

94. The wound dressing of claim 91, wherein the injection port has a tab, and the second adhesive attaches the tab to the second layer.

95. The wound dressing of claim 94, wherein the tab is configured to allow a user to peel the injection port from the distal surface of the second layer.

96. The wound dressing of claim 94, wherein the tab comprises a projection having an inner surface spaced apart from the distal surface of the second layer to allow a user to lift the tab from the distal surface of the second layer.

97. A kit comprising:the wound dressing of claim 91; anda container configured to form or inject the biomaterial.

98. The kit of claim 97, wherein the biomaterial comprises a platelet rich plasma (PRP) or a platelet rich fibrin matrix (PRFM).

99. The kit of claim 97, wherein the container comprises a syringe configured to inject the biomaterial.

100. The kit of claim 97, wherein the container is a centrifuge tube including at least one separator gel configured to form the biomaterial.

101. A method comprising:applying a wound dressing at least partially over a wound; andinjecting a biomaterial through a first opening of a first layer of the wound dressing and a second opening of a second layer of the wound dressing to the wound, wherein air isAttorney Docket No. 005042-00013vented through a plurality of first pores of the first layer and one or more second pores of the second layer.

102. The method of claim 101, wherein the injecting is through a one-way valve of the wound dressing.

103. The method of claim 101, wherein the injecting is through an injection port attached to a distal surface of the second layer.

104. The method of claim 103, further comprising removing the injection port from the distal surface of the second layer after injecting the biomaterial.

105. The method of claim 104, wherein the removing the injection port comprises lifting a tab of the injection port.

106. The method of claim 101, further comprising removing the second layer from the first layer after injecting the biomaterial.

107. The method of claim 101, further comprising coagulating the biomaterial.

108. The method of claim 101, wherein the biomaterial comprises platelet rich plasma (PRP) or platelet rich fibrin matrix (PRFM).

109. The method of claim 101, wherein the air is vented from the second pores directly to the atmosphere.

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