MX2008002958A - Lyophilized platelet rich plasma for the use in wound healing and bone or tissue grafts or repair - Google Patents

Abstract

This invention relates to an improved lyophilized platelet rich plasma used to make a platelet gel wound healant, and methods of preparation and use thereof for healing wounds are disclosed. the improved wound healant comprises therapeutically effective amounts of activated growth factors, platelet ghost, plasma (know as the plasma back bone), white blood cells with optional none, one or more additional anti-oxidant such as vitamin A and/or c and/or E, and/or none one or more antibiotics and/or GHK-Cu.

Description

PLASMA. RICO IN LIOFILIZED PLATELETS FOR USE IN CICATRIZATION OF WOUNDS AND GRAFTING OR REPAIR OF BONE OR TISSUE Field of the Invention The invention relates to an improved lyophilized platelet-rich plasma used for wound healing in platelet gel, and methods of preparing and using it for wound healing are described. The improved wound healing comprises a therapeutically effective amount of activated growth factors with none, one or more optional additional antioxidants such as vitamin A and / or C and / or E, and / or one or more antibiotics and / or GHK-Cu (produced by ProCyte Inc.). The present invention relates to improved lyophilized platelet-rich plasma for use in wound healing and bone or tissue grafts and methods of making the same and using lyophilized or fixed platelets. The present invention generally relates to the therapeutic uses of blood platelets and fresh plasma that has been lyophilized, and most particularly to manipulations or modifications of platelets and plasma, such as in the preparation of freeze-dried compositions that can be rehydrated in time Ref. 191969 application and that when rehydrated they have a normal response to thrombin and other agonists with respect to that of fresh platelets. The compositions of the invention are useful in applications such as wound care.

Antecedents of the Invention Several techniques for platelet conservation have been developed in recent decades. The cryopreservation of platelets with the use of various agents, such as glycerol (Valeri et al., Blood, 43, 131-136, 1974) or dimethyl sulfoxide, "DMSO" (Bock et al., Transfusion, 35, 921- 924, 1995), as the cryoprotector have been made with some success. The best results have been obtained with DMSO. However, a considerable fraction of these cells are partially used after thawing and have the shape of a balloon. These balloon cells do not respond to several agonists, so the overall response of thawed platelets to several agonists is reduced to less than 35% compared to fresh platelets. The shelf life of platelets in cryoconserved DMSO at -80 ° C is reported to be one year, but requires extensive washing and processing to remove cryoprotective agents, and even after the final product has a severe reduction in the ability to form a clot Attempts to dry platelets by lyophilization have been described with paraformaldehyde-fixed platelets (Read et al., Proc. Nati, Acad Sci. USA, 92, 397-401, 1995). Patent of E.U.A. No. 5, 902, 608, issued May 11, 1999, inventors Read et al., Describe and claim a surgical assistant comprising a substrate in which the fixed, dried blood platelets are carried. These dried blood platelets are fixed by contacting the platelets with a fixative such as formaldehyde, paraformaldehyde, cutaraldehyde, or permanganate. The proper functioning of lyophilized platelets that have been fixed by fixative agents in hemostasis is questionable. Spargo et al., Patent of E.U.A. No. 5,736,313, issued April 7, 1998, has described a method in which platelets are loaded overnight with an agent, preferably glucose, and subsequently lyophilized. The platelets are preincubated in a preincubation pH regulator and then loaded with carbohydrate, preferably glucose, having a concentration in the range of about 100 mM to about 1.5M. It is taught that the incubation should be conducted at about 10 ° C to about 37 ° C, most preferably about 25 ° C. , The patent of E.U.A. No. 5,827,741, Beattie et al., Issued October 27, 1998, describes cryoprotectants for human platelets, such as dimethyl sulfoxide and trehalose. Platelets can be suspended, for example, in a solution containing a cryoprotectant at a temperature of about 22 ° C and then cooled to below 15 ° C. This incorporates some cryoprotectant in the cells. Other researchers have sought to load platelets with trehalose through the use of electroporation before drying under vacuum. However, electroporation is very harmful to cell membranes and is thought to activate platelets. Activated platelets have a dubious clinical value. Accordingly, there is a need for the effective and efficient preservation of platelets in such a way that they maintain, or retain, their biological properties, particularly their response to platelet agonists such as thrombin, and that they release their growth factors. In addition, it would also be useful to expand the types of present vehicles that are useful for wound care. There have been many different substances and methods developed in the past to treat wounds, depending on the type and location and severity of the wound. A wound is usually defined as an injury to an area of the body of a human or animal. Although the injury to the surface of the skin is the best known type of wound, the surfaces of internal organs can also be injured, such as during surgery, rupture of the spleen or liver, or as a result of trauma to the body surface in the neighborhood of an internal organ. The medical practice characterizes wounds as chronic or acute, in accordance with the persistence and severity of the wound. A chronic wound is one that is prolonged or slow to heal, and that does not heal quickly. An acute wound is one that occurs relatively quickly, and heal also relatively quickly. Tissue wounds can have a wide spectrum of manifestations, as small as simply an abnormal microscopic tear or fissure in the tissue (or a surface thereof), or as large as abrasion or ablation of the skin covering a substantial portion of the tissue. body, such as in a burn victim. Acute wounds that cover a large, mobile surface are usually the most difficult to protect from infection, and to heal. Blood and body fluids include several substances that affect wound healing. Blood is the main means to deliver healing agents to the wound site, and to transport extraneous or dangerous substances away from the wound.

Whole blood is composed mainly of three main types of cells suspended in a protein-rich solution known as plasma. The three main cell types in whole blood are erythrocytes (a.k.a. red blood cells), leukocytes (a.k.a. white blood cells) and thrombocytes (a.k.a. platelets). Red blood cells are iron-containing cells that facilitate transport and transfer oxygen to body tissue, and the removal of carbon dioxide. White blood cells perform a variety of functions such as phagocytosis of foreign bodies and production of antibodies, and are primarily responsible for combating infections and foreign substances within the blood or site of the wound. Platelets perform many functions such as plugging leaks in blood vessels and help initiate the process that leads to the formation of a blood clot; Platelets contain substances known as growth factors that facilitate the formation of new tissue. Although there are several methods for separating whole blood into its various components, one of the most convenient and rapid methods is achieved by differential centrifugation of the blood or some of its components (ie, apheresis). By using apheresis, red and white blood cells and plasma can be separated and returned to the donor or patient's body, which leaves the platelets sequestered in an essentially concentrated form for use in wound healing techniques. This can be done at a blood collection center, blood bank, doctor's clinic or hospital. From blood drawn from a patient or from other forms of collection methods, platelets can therefore be obtained and activated for use in the patient; methods of using the patient's own blood are called "autologous" or "autogenic" donor methods. Another method that uses blood donated by one or more third parties to be used by a patient is called "homologous" or "heterologous" donor methods, or collectively called "allogenic" methods. One of the proteins suspended in plasma is fibrinogen, which reacts with substances released in (or attracted to) wound sites to produce sticky fibrin filaments. Such reactions result in the cross-linking of the fibrin filaments to form a mesh that supports and supports the deposition or growth of other tissue materials at the site of the wound. Therefore, the need for fresh plasma is also known in the art as "the plasma spine". The process of wound healing is generally considered to occur in several stages, generally known as the healing cascade. After tissue injury, platelets are among the first cells that appear in the vicinity of the wound. Activation of a platelet by an agonist such as thrombin, or other agonists such as those listed elsewhere herein, leads to the release of granule material from the interior of the platelet. The activation of granulation results in the release of proteins known as growth factors, mainly concentrated in the alpha granules of platelets. These factors of c have been known that when applied to wounds, growth factors increase the rate of collagen deposition, vascular internal growth, fibroblast proliferation and overall healing. The release of a protein known as platelet-derived growth factor (PDGF) is a chemotactic signal for monocytes, neutrophils and fibroblasts, which then move toward the wound, to initiate the inflammatory stage of the wound healing process. During this time, monocytes secrete a number of factors including PDGF and transform beta-1 growth factor (also found in platelets), which recruits and activates fibroblasts, to begin the repair stage of the healing process. Subsequently, wound healing continues through the process of collagen remodeling within the wound. Therefore, the importance of the capacity of lyophilized platelet-rich plasma to release its growth factors.

Based on the above general scientific principles, wound sealants made of biological materials obtained mainly from tissue other than blood platelets are also known in the field. One example is wound sealants such as "fibrin glue", which are often essentially a mixture of co-coagulants (thrombin and calcium), concentrated fibrinogen and other coagulation proteins. In most applications, the main roles of fibrin glue are to seal wound surfaces to prevent loss of blood and other body fluids after surgery, and to provide adhesion between adjacent tissue surfaces. These products form a hard, skin-like covering over the area to be sealed, and tend to yield to the movement of the limbs. Although there has been much research concerning fibrin glue, this material belongs to a separate field of the present invention, mainly because fibrin glues typically contain cryoprecipitate proteins without platelets. The use of fibrin glue is extensively described in the scientific literature; for example, see the references cited in the US patent. No. 5,585,007 issued to Antanavich et al on December 17, 1996. Compositions for treating wounds derived from platelet-enriched concentrates are known and have certain advantages over non-platelet materials such as fibrin glue. One reason for this is that natural wound healing agents are released by platelets. In addition, the concentration of platelets also allows a concentrated amount of wound healing factors. Representative examples of compositions for treatment of wounds derived from platelets are described, for example, in Hood patent of E.U.A. No. 5,733,545; Knighton patent of E.U.A. No. 5,165,938; and Gordiner patent of E.U.A. No. 5,599,558. This form of treatment has major problems, such as time consuming and blood collection problems sometimes with expensive machines and disposable materials. Platelet concentrates are typically isolated by the differential centrifugation procedure, which essentially allows blood to be separated into at least three different components: packed erythrocytes (red blood cells), plasma and platelet concentrate. The platelet concentrate can be combined with a solution of either sodium or calcium mixed with thrombin ("calcified thrombin"), which instantly form a composition of activated platelets which, when made with the necessary viscosity, can be used as. ' a wound sealer. The chemical reactions and cascades that normally occur when thrombin is added to concentrated platelets are indeed complex. See, for example, Reeder, et al, in Proceedings of the American Academy of Cardiovascular Perfusion, Vol. 14, January 1993. Such wound sealants are typically fixed in a hard mass that covers the site of application, so that Seal the site against additional blood loss and external contaminants. There are a number of drawbacks associated with conventional wound compositions derived from platelet concentrates. For example, platelet activation leads to instantaneous hardening of the material and therefore requires the physician to activate and apply the platelet composition to the wound site within seconds of activation. Also, certain platelet compositions must be applied to the wound site on a daily basis and therefore require regular blood draw from the patient. This consumes time and if more of the compositions are needed, then another needle and the procedure time required to make the composition are needed. Accordingly, an improved, lyophilized platelet-enriched plasma treatment composition would be desirable to avoid or diminish the problems associated with wound compositions enriched with typical platelets.

SUMMARY OF THE INVENTION In one aspect of the present invention, there is provided a dehydrated composition comprising freeze dried platelets and fresh plasma which are effective in preserving biological properties during freeze drying and rehydration. These platelets are rehydratable to have a normal response to at least one agonist, such as thrombin. For example, substantially all freeze-dried platelets of the invention, when rehydrated and mixed with thrombin (1 U / ml) form a clot within three minutes at 37 degrees centigrade. The dehydrated composition may include one or more other agents, such as antibiotics, antifungals, growth factors or the like, according to the desired therapeutic application. The present invention relates to an improved lyophilized platelet and plasma composition for the treatment of wounds, a method for making and using same. The composition comprises lyophilized platelets and plasma composition in liquid form at the wound site and will prevent the material from flowing away from the site. Optional antibiotics can be included in the improved composition to prevent infections at the wound site. The presence of the antioxidant, which includes vitamins and antioxidants that are not vitamins, and other healing promotion materials that do not decrease, substantially interfere with or even destroy the different reactions of thrombin activation. The lyophilized platelet gels of the invention containing fresh plasma are prepared for topical application at the wound site and avoid the requirement of daily reapplication. Methods for making and using embodiments of the invention are also described. One such method is a process for preparing a dehydrated composition comprising providing a source of platelets and fresh plasma and lyophilizing the platelets and plasma. The rehydration is preferably done with deionized water. Although the composition of the invention is preferably used for topical application to the exterior surface of chronic wounds such as foot ulcers of diabetics, the composition can be applied to facilitate the healing of other wounds such as surgery and burns. However, the composition of the invention and the methods described herein are not limited to topical application only. The composition of the invention increases the amount of growth factors in the wound and thus facilitates the promotion of the healing rate. This may be especially important in patients whose "wounds", especially those with chronic wounds that may lack sufficient circulation to facilitate the healing cascade. The invention described here also facilitates coverage of the wound area with a substance that prevents or helps reduce the infection caused by most bacteria. The practice of the invention allows the manipulation or modification of platelets and plasma while maintaining, or preserving, the biological properties, such as a response to thrombin. The freeze-dried platelets of the invention and the plasma that includes the freeze-dried platelets are substantially shelf-stable at different temperatures when packaged in moisture barrier materials. In more general terms, the invention described herein expands the uses for concentrated platelet materials, especially those in gel form, by improving the speed and convenience of making the composition; The invention described herein also improves the performance of the concentrated platelet composition by making it more usable for applications for prolonged periods, and by improving the wound healing properties and fighting the infection. Another aspect of the present invention involves adding one or more antibiotic substances in one or more times during the processing period, so that the resulting platelet and plasma concentrate composition contains either one or a variety of the antibiotics. The use of an antibiotic in concentrated platelet and plasma compositions that increase the complex healing cascade is indeed novel. The invention described herein involves adding those substances in a manner that does not decrease, substantially interfere with or even destroy these different reactions, pH and potency equilibria. Another aspect of the present invention involves adding one or more vitamins to the concentrated platelet gel. It is known that vitamins have wound healing and antioxidant properties. Representative examples of suitable vitamins, but in no way limiting, include vitamin E, vitamin A, vitamin C and other retinoids. In another aspect of the invention, antioxidants that are not vitamins can be included in the concentrated platelet gel. Representative non-limiting examples of those antioxidants include beta-carotene.

Detailed Description of the Invention For purposes of simplicity and to give the claims of this patent application the broadest interpretation and possible construction, the following definitions will apply: (a) The phrase blood collection or blood draw (or similar phrase) ) includes techniques, materials and apparatuses known in the field, such as (for example) the inclusion of anticoagulation materials, the use of extraction apparatus and blood infusion. (b) The phrase growth factor means any material (s) that promotes the growth of a weave. (c) The term thrombin may include calcified thrombin, in particular, about 5,000 units of thrombin per 5 ml of 10% aqueous solution of calcium chloride; it may include calcified bovine thrombin as well as autologous thrombin, allogeneic thrombin or recombinant human thrombin. (d) The term viscosity means those characteristics of the specified material (s) that determine the degree of gelation, such as (for example) the firmness or hardness of the material, or the degree to which the material resists flow as a fluid. (e) The term "therapeutically effective amount" means the quantity or amounts of the constituent elements or combination thereof necessary to increase wound healing such as,. for example, the reduction in the volume of surface area of a wound, the increase in the amount of granulation tissue or other biological material that facilitates collagen deposition, vascular internal growth, proliferation of fibroblasts or global healing; it is assumed that all versions of the invention described herein have the therapeutically effective amount (s) of constituent substances, or combinations thereof. (f) The term "antioxidant" refers to any material (s) that have (n) antioxidant properties. Antioxidant would include, without limitation, vitamins such as vitamins C, A and E and not vitamins such as -carotene. Also for purposes of simplicity, the conjunction "and" may also be adopted to include the dilemma "or" and vice versa, whenever necessary to give the claims of this patent application the widest interpretation and possible construction. Likewise, when the plural form is used, it can be adopted to include the singular forms and vice versa. In the most general terms, the invention includes a wound healing composition comprising activated growth factors and ascorbic acid. In the predominant version of the invention, the growth factors are included within the platelets. The body produces many substances generally known as growth factors, and those growth factors are contemplated for use in the present invention. Preferred growth factors for use in the present invention are selected from the group consisting of platelet-derived growth factor (PDGF), platelet-derived angiogenesis factor (PDAF), vascular endothelial growth factor (VEGF), growth factor. epidermal platelet-derived (PDEGF), platelet factor 4 (PF-4), transforming growth factor .beta. (TGF-B), acid fibroblast growth factor (FGF-A), basic fibroblast growth factor (FGF-B), transforming growth factor alpha (TGF-A), growth factors similar to insulin 1 and 2 (IGF-1 and IGF-2), proteins related to beta-thromboglobulin (BTG), thrombospondin (TSP), fibronectin, von Wallinbrand factor (v F), fibropeptide A, fibrinogen, albumin, plasminogen activator inhibitor 1 (PAI-1), osteonectin, normal T cells regulated under activation expressed and supposedly secreted (RANTES), gro-alpha, vitronectin, D-fibrin dimer, factor V, antithrombin III, immunoglobulin-G (IgG), immunoglobulin- ( IgM), immunoglobulin-A (IgA), a2-macroglobulin, angiogenin, Fg-D, elastase, keratinocyte growth factor (KGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), tumor necrosis factor (TNF), growth factor of fibroblasts (FGF) and interleukin-1 (IL-1), keratinocyte growth factor-2 (KGF-2), and combinations thereof. One of the important common characteristics for each substance, which supports the inclusion of each in this particular group, is that each of these substances is known or known to increase the growth of the cells or tissue. Moreover, these substances, or various combinations thereof, are known or believed to work together in an unexpected synergistic manner to promote wound healing. Suitable, non-limiting antioxidants useful in the invention include but are not limited to vitamins such as vitamin C (ascorbic acid), vitamin E, vitamin A and other retinoids; and carotenes such as beta-carotene. In the practice of this invention, ascorbic acid as an antioxidant is particularly preferred. Platelets are separated from the red blood cells and white blood cells of whole blood, mainly through differential centrifugation, although any other method suitable for separating platelets from whole blood can be used in the practice of this invention. The overall composition of the invention described herein may contain incidental amounts of white blood cells, due to the fact that platelets are seldom totally isolated from the other components of the blood. It is believed that the present invention contains only trace amounts of white blood cells; it is believed that the white blood cell count of the present invention will typically be below about 3 times 10 ~ 7 cells / ml. The bioactive material in the invention is almost exclusively platelets. The average platelet volume range of the platelets that are sequestered is in the range of about 6.6 to 8.4 femtoliters, with an average of about 7.7 femtoliters; this may indicate that the platelets that are sequestered are relatively larger or younger than the overall population of platelets. Activation of growth factors can occur in a variety of ways, by a variety of substances such as activators or agonists. In the invention described herein, that activation results from lysine and the inclusion of an activator or agonist selected from the group consisting of thrombin, glass, collagen, serotonin, adenosine diphosphate (ADP) and acetylcholine (ACH), and combinations thereof . In a particular and preferred version of the invention, growth factors are included within concentrated platelets, and that activation results from the inclusion of thrombin. One of the important common characteristics for each substance, which supports the inclusion of each in this particular group, is that each substance is known or believed to increase cell or tissue growth in addition to the ability to activate platelets. Moreover, these substances, or various combinations thereof, are known or believed to work together in an unexpected synergistic manner to promote wound healing. The activator or agonist added to the platelet and plasma concentrate is in an amount sufficient to facilitate the formation of the clot (gel) having a predetermined viscosity while sufficiently activating the growth factors present in the composition. In the preferred case where thrombin is used as the activator to produce a final soft gel wound composition in a soft gel form, the amount of thrombin generally ranges from about 100 U to about 10,000 U, preferably about 900 U and about 1100 U, most preferably 1000 U per 10 cc of platelet concentrate. Thrombin is available as Thrombogen-RTM-thrombin, topical USP (bovine origin) in bottles containing 5000 thrombin units (Johnson &Johnson Medical Inc., Arlington, Tex., E.U.A). Since the mixture of thrombin or other agonists will activate the growth factors, thrombin (or other agonists / activators) usually must be the last substance to be mixed immediately before the desired gelatinous state is established.

In addition to a method for making a wound healing composition, the invention described herein may also include a method of treating wounds, comprising the steps of applying a sufficient amount of a composition of material comprising growth factors to increase wound healing. the wound. That method of treating a wound may include the use of any of the compositions described herein; it may also include the use of any composition made by any of the methods described herein. Once applied to the wound, the composition can remain on the wound as much as 5 days, and perhaps more depending on the circumstances such as the location of the wound and other wound characteristics. Although the composition and method described herein are especially useful for the treatment of chronic wounds, they can also be used in the treatment of acute wounds. Example 1 Preparation of lyophilized platelet-rich plasma for use in wound healing (chronic or acute) and bone or tissue grafts (a) The first method for blood components obtained from a blood bank or collection center: The components needed: Platelets in stock or from a single donor (containing at least 5 x 10 to 9 th platelets) approximately 40 ml to 50 ml per bag. Fresh frozen plasma in stock or from a single donor (approximately 250 ml) per bag. Centrifuge with the capabilities of turning tubes from 50 ml to 5000 rpm. Tubes of 50 ml Pipettes of .01 ul to 50 ml Bottles with caps and caps.

Method: Remove the platelets from the bag and place in a 50 ml tube and centrifuge for 15-20 min at 5000 rpm to form a platelet plug, which is known in the art. Remove and discard the platelet deficient plasma from the platelet tube. Thaw fresh frozen plasma and insert a quantity into the container with platelet stopper to produce a platelet count of between 250,000 to 4,500,000 platelets per milliliter. Take care that the fresh plasma does not remain thawed for more than 4 hours. Gently rotate back and forth to make platelets and plasma mix well. Pipet the desired amount of PRP into the bottles to be lyophilized and place the plugs in place. Lyophilize once (be careful not to allow any heating to occur). The first cycle should be 48 hours. The second cycle should be 12 hours. For this example, 10 ml of PRP was lyophilized and capped. To apply to the wound by using 10 ml bottles of LPRP: Rehydrate by using deionized water (10 ml) and let stand for at least 10 to 15 minutes. Remove the LPRP by using a syringe of mi. Add 1 ml of thrombin (bovine) lOOOu per milliliter. Place on the wound and cover with a wet gauze. Cover the wound with an exclusive bandage. Let stand in place for 4 to 7 days without changing the bandage. Repeat if necessary. Example 2 (a) The second method for blood components obtained from a blood bank or collection center: components required platelets in pool or from a single donor (containing at least 5 x 10 to 9a platelets) 40 mi to 50 mi per bag. Fresh frozen plasma in stock or from a single donor (approximately 250 ml) per bag. Centrifuge with the capabilities of turning tubes from 50 ml to 5000 rpm. Tubes of 50 ml Pipettes of .01 ul to 50 ml Bottles with caps and caps. GHK-Cu varies from .02 mg / ml to .5 mg / ml in liquid form Method: Remove the platelets from the bag and place in a 50 ml tube and centrifuge for 15-20 min at 5000 rpm to form a platelet plug, which is known in the art. Remove and discard the platelet deficient plasma from the platelet tube. Thaw frozen fresh plasma and insert a quantity into the container with platelet stopper to produce a platelet count of between 250, 000 to 4,500,000 platelets per milliliter. Take care that the fresh plasma does not remain thawed for more than 4 hours. Add a quantity of GHK-Cu to equal 1 mL per 9 mL of LPRP. Gently rotate back and forth to make platelets and plasma mix well. Pipet the desired amount of PRP into the bottles to be lyophilized and place the plugs in place. Lyophilize once (be careful not to allow any heating to occur). The first cycle should be 48 hours. The second cycle should be 12 hours. For this example, 10 ml of PRP was lyophilized and capped. To apply to the wound by using 10 ml bottles of LPRP: Rehydrate by using deionized water (10 ml) and let stand for at least 10 to 15 minutes. Remove the LPRP by using a syringe of mi. Add 1 ml of thrombin (bovine) lOOOu per milliliter. Place on the wound and cover with a wet gauze. Cover the wound with an exclusive bandage. Let stand in place for 4 to 7 days without changing the bandage. Repeat if necessary. (b) The third method for blood components obtained from a blood bank or collection center: The components needed: Platelets in stock or from a single donor (containing at least 5 x 10 to the 9th platelets) 40 mi to 50 mi per bag. Fresh frozen plasma in stock or from a single donor (approximately 250 ml) per bag. Centrifuge with the capabilities of spinning tubes 50 ml up to 5000 rpm. Tubes of 50 ml Pipettes of .01 ul to 50 ml Bottles with caps and caps. GHK-Cu varies from .02 mg / ml to .5 mg / ml gauze. Method: Remove the platelets from the bag and place in a 50 ml tube and centrifuge for 15-20 min at 5000 rpm to form a platelet plug, which is known in the art. Remove and discard the platelet deficient plasma from the platelet tube. Thaw fresh frozen plasma and insert a quantity into the container with platelet stopper to produce a platelet count of between 250,000 to 4,500,000 platelets per milliliter. Take care that the fresh plasma does not remain thawed for more than 4 hours. Gently rotate back and forth to make platelets and plasma mix well. Pipet the desired amount of PRP into the bottles to be lyophilized and place the plugs in place. Lyophilize once (be careful not to allow any heating to occur). The first cycle should be 48 hours. The second cycle should be 12 hours. For this example, 10 ml of PRP was lyophilized and capped. To apply to the wound by using 10 ml bottles of LPRP: Rehydrate by using deionized water (10 ml) and let stand for at least 10 to 15 minutes. Remove the LPRP by using a syringe of mi. Add 1 ml of thrombin (bovine) lOOOu per milliliter. Place on the wound and cover with gauze with GHK-Cu. Cover the wound with an exclusive bandage. Let stand in place for 4 to 7 days without changing the bandage. Repeat if necessary. (c) the fourth method for blood components obtained from a blood bank or collection center: The components needed: Platelets in stock or from a single donor (containing at least 5 x 10 to the 9th platelets) 40 mi to 50 mi per bag. Fresh frozen plasma in stock or from a single donor (approximately 250 ml) per bag. Centrifuge with the capabilities of spinning tubes 50 ml up to 5000 rpm. Tubes of 50 ml Pipettes of .01 ul to 50 ml Bottles with caps and caps. (Vitamin C is an amount equal to 10%) Method: Remove the platelets from the bag and place in a 50 ml tube and centrifuge for 15-20 min at 5000 rpm to form a platelet plug, which is known in the art . Remove and discard the platelet deficient plasma from the platelet tube. Thaw fresh frozen plasma and insert a quantity into the container with platelet stopper to produce a platelet count of between 250,000 to 4,500,000 platelets per milliliter. Take care that the fresh plasma does not remain thawed for more than 4 hours. Add an amount of vitamin C that is equal to 1 ml per 9 ml of LPRP. Gently rotate back and forth to make platelets and plasma mix well. Pipet the desired amount of PRP into the bottles to be lyophilized and place the plugs in place. Lyophilize once (be careful not to allow any heating to occur). The first cycle should be 48 hours. The second cycle should be 12 hours. For this example, 10 ml of PRP was lyophilized and capped. To apply to the wound by using 10 ml bottles of LPRP: Rehydrate by using deionized water (10 ml) and let stand for at least 10 to 15 minutes. Remove the LPRP by using a syringe of mi. Add 1 ml of thrombin (bovine) lOOOu per milliliter. Place on the wound and cover with gauze with GHK-Cu. Cover the wound with an exclusive bandage. Let stand in place for 4 to 7 days without changing the bandage. Repeat if necessary. (d) the fifth method for blood components obtained from a blood bank or collection center: The components required: Platelets in stock or from a single donor (containing at least 5 x 10 to 9 th platelets) 40 mi to 50 mi per bag. Fresh frozen plasma in stock or from a single donor (approximately 250 ml) per bag. Centrifuge with the capabilities of turning tubes from 50 ml to 5000 rpm. Tubes of 50 ml Pipettes of .01 ul to 50 ml Bottles with caps and caps. (Vitamin C is an amount equal to 10%) Method: Remove the platelets from the bag and place in a 50 ml tube and centrifuge for 15-20 min at 5000 rpm to form a platelet plug, which is known in the art. Remove and discard the platelet deficient plasma from the platelet tube. Thaw fresh frozen plasma and insert a quantity into the container with platelet stopper to produce a platelet count of between 250,000 to 4,500,000 platelets per milliliter. Take care that the fresh plasma does not remain thawed for more than 4 hours. Gently rotate back and forth to make platelets and plasma mix well. Pipet the desired amount of PRP into the bottles to be lyophilized and place the plugs in place. Lyophilize once (be careful not to allow any heating to occur). The first cycle should be 48 hours. The second cycle should be 12 hours. For this example, 10 ml of PRP was lyophilized and capped. To apply to the wound by using 10 ml bottles of LPRP: Rehydrate by using deionized water (10 ml) and let stand for at least 10 to 15 minutes. Remove the LPRP by using a syringe of mi. Add 1 ml of thrombin (bovine) lOOOu per milliliter. Add 1 ml of 10% vitamin C, A.K.A (ascorbic acid). Place on the wound and cover with gauze with GHK-Cu. Cover the wound with an exclusive bandage. Let stand in place for 4 to 7 days without changing the bandage. Repeat if necessary. (e) the sixth method for blood components obtained from a blood bank or collection center: The components required: Platelets in stock or from a single donor (containing at least 5 x 10 to 9 th platelets) 40 mi to 50 mi per bag. Fresh frozen plasma in stock or from a single donor (approximately 250 ml) per bag. Centrifuge with the capabilities of turning tubes from 50 ml to 5000 rpm. Tubes of 50 ml Pipettes of .01 ul to 50 ml Bottles with caps and caps. (Vitamin C is an amount equal to 10%) GHK-Cu varies from .02 mg / ml to .5 mg / ml in liquid form. Method: Remove the platelets from the bag and place in a 50 ml tube and centrifuge for 15-20 min at 5000 rpm to form a platelet plug, which is known in the art. Remove and discard the platelet deficient plasma from the platelet tube. Thaw fresh frozen plasma and insert a quantity into the container with platelet stopper to produce a platelet count of between 250,000 to 4,500,000 platelets per milliliter. Take care that the fresh plasma does not remain thawed for more than 4 hours. Add an amount of vitamin C equal to .05 mi to 1 ml per 9 ml of LPRP Add a quantity of GHK-Cu equal to .05 ml at 1 ml per 9 ml of LPRP. Gently rotate back and forth to make platelets and plasma mix well. Pipet the desired amount of PRP into the bottles to be lyophilized and place the plugs in place. Lyophilize once (be careful not to allow any heating to occur). The first cycle should be 48 hours. The second cycle should be 12 hours. For this example, 10 ml of PRP was lyophilized and capped. To apply to the wound by using 10 ml bottles of LPRP: Rehydrate by using deionized water (10 ml) and let stand for at least 10 to 15 minutes. Remove the LPRP by using a syringe of mi. Add 1 ml of thrombin (bovine) lOOOu per milliliter. Add 1 ml of 10% vitamin C, A.K.A (ascorbic acid). Place on the wound and cover with gauze with GHK-Cu. Cover the wound with an exclusive bandage. Let stand in place for 4 to 7 days without changing the bandage. Repeat if necessary.

Example 3 (a) The sixth method for components of human or animal blood, obtained from apheresis either autologous or homologous: The components needed: Platelets from a single donor (obtained from apheresis) approximately 40 ml to 50 ml per bag. Centrifuge with the capabilities of turning tubes from 50 ml to 5000 rpm. Tubes of 50 ml Pipettes of .01 ul to 50 ml Bottles with caps and caps.

Method: Remove platelets from apheresis platelets. Add fresh plasma into the platelets in an amount to cause a platelet count of between 250,000 to 4,500,000 platelets per milliliter. Be careful that fresh platelet-rich plasma does not remain thawed for more than 6 hours. Gently rotate back and forth to make platelets and plasma mix well. Pipet the desired amount of PRP into the bottles to be lyophilized and place the plugs in place. Lyophilize once (be careful not to allow any heating to occur). The first cycle should be 48 hours. The second cycle should be 12 hours. For this example, 10 ml of PRP was lyophilized and capped. To apply to the wound by using 10 ml bottles of LPRP: a. Rehydrate by using deionized water (10 ml) and let stand for at least 10 to 15 minutes. b. Remove the LPRP by using a 20 ml syringe. c. Add 1 ml of thrombin (bovine) lOOOu per milliliter. d. Place on the wound and cover with a wet gauze. and. Cover the wound with an exclusive bandage. F. Let stand in place for 4 to 7 days without changing the bandage. g. Repeat if necessary.

Example 4 (c) The third method for components of human blood obtained from a blood bank and activated prior to lyophilization with thrombin (bovine or intrinsic): The components required: Platelets in stock or from a single donor (containing at least minus 5 x 10 to the 9th platelets) approximately 40 ml to 50 ml per bag. Fresh frozen plasma in stock or from a single donor (approximately 250 ml) per bag. Centrifuge with the capabilities of turning tubes from 50 ml to 5000 rpm. Tubes of 50 ml Pipettes of .01 ul to 50 ml Bottles with caps and caps. 6. Thrombin and calcium chloride or spheres of glass or glass wool Method: Remove the platelets from the bag and place in a 50 ml tube and centrifuge for 15-20 min at 5000 rpm to form a platelet plug, which is known in the art. Remove and discard the platelet deficient plasma from the platelet tube. Remove 50% of the platelet plug and activate the cap with thrombin or pass the platelets through either glass wool or glass beads and allow the activated platelets to rest for approximately 15 minutes, then centrifuge for 15 minutes and remove the serum without the stopper. After Pipetting equal amounts of the serum into the bottles in equal amounts. Thaw fresh frozen plasma and insert a quantity into the container with platelet stopper to produce a platelet count of between 250,000 to 4,500,000 platelets per milliliter. Take care that the fresh plasma does not remain thawed for more than 4 hours. Gently rotate back and forth to make platelets and plasma mix well.

Pipet the desired amount of PRP into the bottles to be lyophilized and place the plugs in place. Lyophilize once (be careful not to allow any heating to occur). The first cycle should be 48 hours. The second cycle should be 12 hours. For this example, 10 ml of PRP was lyophilized and capped. To apply to the wound by using bottles of 10 ml of ALPRP: Rehydrate by using deionized water (10 ml) and let it rest for at least 10 to 15 minutes. Remove the LPRP by using a syringe of mi. Place on the wound and cover with a wet gauze. Cover the wound with an exclusive bandage. Let stand in place for 4 to 7 days without changing the bandage. Repeat if necessary. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (28)

1. Claims Having described the invention as above, the content of the following claims is claimed as property. A method for preparing platelet-rich plasma to obtain growth factors for use in wound healing, characterized in that it comprises: providing platelets selected from a source of platelets from the pool and from a platelet source from a single donor; concentrate the platelets in a platelet-rich plasma; discard the platelet-deficient plasma from platelet-rich plasma after concentrating the platelet-rich plasma; add fresh plasma in the platelet-rich plasma to produce a count between 250,000 and 4,500,000 per milliliter; mix the platelet-rich plasma with the fresh plasma to provide a plasma rich in platelets; and treating the platelet-rich plasma mixture in non-chemical form immediately after mixing to release growth factors from platelet-rich plasma.
2. 2. The method according to claim 1, characterized in that the non-chemical treatment includes the lyophilization step of the platelet-rich plasma mixture to provide a lyophilized platelet-rich plasma.
3. 3. The method according to claim 2, characterized in that it includes the step of freezing the mixture of platelet-rich plasma at about -50 ° C for about 4 hours and then raising to a temperature of about -20 ° C for the duration of the treatment. lyophilization procedure.
4. 4. The method according to claim 2, characterized in that it includes the step of rehydrating the lyophilized platelet-rich plasma with the use of deionized water.
5. The method according to claim 4, characterized in that it includes the step of rehydrating the platelet-rich plasma for a period of 10-15 minutes.
6. 6. The method according to claim 4, characterized in that it includes the step of recovering lyophilized platelet-rich plasma after rehydration.
7. The method according to claim 6, characterized in that it includes the step of activating the lyophilized platelet-rich plasma to be placed on a wound.
8. The method according to claim 7, characterized in that it includes the step of adding thrombin to the lyophilized platelet-rich plasma.
9. 9. The method according to claim 8, characterized in that it includes the step of mixing the thrombin, mixing at least one antibiotic in sufficient quantity (s) to reduce infection by bacteria.
10. The method according to claim 9, characterized in that it includes the step of selecting the antibiotic from the group consisting of an antibiotic which is at least bactericidal for Pseudomonas and Klebsella bacteria.
11. The method according to claim 10, characterized in that it includes the step of selecting the antibiotic from the group consisting of neosporin, vancomycin and gentamicin, and combinations thereof.
12. The method according to claim 2, characterized in that it includes the step, either before or after lyophilization of the platelet rich plasma mixture, of adding at least one retinoid in sufficient amount (s) to improve wound healing.
13. The method according to claim 2, characterized in that it includes the step, either before or after lyophilization, mixing in a therapeutically effective amount (s), concentrated platelets, ascorbic acid, at least one retinoid and at least one antibiotic bactericide to at least Pseudomonas and Klebsella bacteria.
14. 14. A method for obtaining growth factors for use in wound healing, characterized in that it comprises treating a growth factor starting material in non-chemical form to release growth factors from the growth factor starting material.
15. 15. The method according to claim 14, characterized in that the growth factor starting material is selected from the group consisting of platelets and platelet-rich plasma, and combinations thereof.
16. 16. The method according to claim 14, characterized in that the non-chemical treatment includes the freeze-drying step of the growth factor starting material.
17. 17. The method according to claim 14, characterized in that it includes the step of recovering growth factors of the treated growth factor starting material.
18. 18. The method according to claim 17, characterized in that it includes the step of rehydrating the growth factor starting material to recover the growth factors.
19. 19. A composition of human growth factor in freeze-dried stock prepared by lyophilizing a starting material of human growth factor in stock in a chemical-free manner.
20. The lyophilized human growth factor composition according to claim 19, characterized in that the growth factor starting material is selected from the group consisting of platelets, platelet-rich plasma, and combinations thereof.
21. 21. The composition of human growth factor lyophilized according to claim 19, characterized in that the composition is lyophilized at a temperature of -20 ° C or lower.
22. 22. The lyophilized human growth factor composition according to claim 19, characterized in that the growth factor starting material is mixed with fresh plasma before lyophilization.
23. 23. The composition of human growth factor in lyophilized stock in accordance with the claim 22, characterized in that the starting material of growth factor is immediately lyophilized after mixing with the fresh plasma.
24. 24. The composition of human growth factor in lyophilized stock in accordance with the claim 23, characterized in that substantially no heating occurs during mixing with the fresh plasma before lyophilization.
25. 25. The lyophilized human growth factor composition according to claim 23, characterized in that the growth factors are selected from the group consisting of PDGF, PDAF, PD, VEGF, PDEGF, PF-4, TGF-B, BTG, TSP, vWF, PAI-1, RANTES, IgG, Ig, IgA, FgD, KGF, EGF, FGF, FNT, IL-1, KGF-2, IGF-1 and IGF-2
26. 26. The factor composition of Human growth in lyophilized pool according to claim 19, characterized in that it comprises a pharmaceutically acceptable vehicle.
27. 27. The composition of human growth factor lyophilized according to claim 26, characterized in that the vehicle is a gel.
28. 28. The composition of human growth factor lyophilized according to claim 19, characterized in that it is carried on a bandage to be applied to a wound. Resurrection of the Invention This invention relates to an improved lyophilized platelet-rich plasma used to make a wound healing in platelet gel, and methods of preparation and use thereof for wound healing are described. The improved wound healing comprises a therapeutically effective amount of activated growth factors, phantom platelets, plasma (known as the backbone of the plasma), white blood cells with none, one or more optional additional antioxidants such as vitamin A and / or C and / or E, and / or one or more antibiotics and / or GHK-Cu.