ES2262444B1 - USE OF PLASMA RICH IN PLATES FOR THE PREPARATION OF A MEDICINAL PRODUCT TO COMBAT FRIBROSIS. - Google Patents

Abstract

Use of platelet rich plasma for the preparation of a drug to fight fibrosis. The platelet-rich plasma (PRP) of the invention has a platelet count greater than 300,000 platelets / ml and is obtained by known procedures. Medications comprising PRP are preferably used in tissue repair processes, more preferably tendons. Medications comprising PRP are also used in tissue repair processes secondary to surgical processes. Finally, medicines that comprise PRP are used in the treatment of diseases that are associated with associated fibrosis, such as cystic fibrosis, chronic liver damage or progressive fibrosis of the palmar fascia, as well as in the prevention of the development of fibrosis associated with The mentioned diseases.

Description

Use of platelet rich plasma for Preparation of a medicine to fight fibrosis.

Field of the Invention

The invention relates to the use of platelet rich plasma (PRP) for the preparation of medicaments intended to prevent or decrease the development of fibrosis during regenerative processes, especially in the case of tendons or regenerative processes secondary to surgical operations, and during pathological processes. such as diseases that normally occur with associated fibrosis. Additionally, the invention also relates to the use of platelet-rich plasma for the preparation of medicaments intended to combat established fibrosis, as in chronic liver damage, progressive fibrosis of the palmar fascia or cystic fibrosis already disengaged.
rolled.

State of the art

From collaboration between disciplines have emerged spectacular progress in recent years. get app practical clinics derived from these biological investigations and Molecular is one of the challenges we now face. Among these developments, the advances in the study of  molecular mechanisms that control cell signaling and lead to the regeneration of connective tissues (15).

Proteins are directly responsible for state and cell destinies in the different pathological situations or traumatological; Within this biological context are the growth factors, which are proteins that control the evolution of the regeneration and repair processes of different fabrics

Growth factors are proteins that are found naturally in the body and act as signaling agents for cells. Its function is part of a wide network of cellular communications that influence critical cellular functions such as division, matrix synthesis and tissue differentiation (10, 20).

Growth factors can have three Possible types of functions:

one.

Autocrine : in which growth factors influence the cell of origin, or another cell of the same phenotype as it.

2.

Paracrine : in which growth factors influence adjacent or neighboring cells, which are different in phenotype from the cell of origin.

3.

Endocrine : in which the growth factors influence cells of different phenotypes with respect to the cells of origin and which are located anatomically at a distance.

Therefore growth factors can have effects on multiple cell types and can induce a set of cellular functions in a variety of tissues.

Once the origin cells are activated, you are They release growth factors in a given region. To the activated these cells can both activate the secretion of factors, previously formed and stored for example in vesicles, like the synthesis of these. The factors join specific cell receptors in target cells. This type of interactions are very specific, can be a very reaction simple, joining a factor to a receiver, or more complex if they join several factors to different receptors causing various answers. In this biological system several forms of the same factor they can join the same receiver, and a simple factor can join several receivers (10, 20).

Recent studies (6, 8, 11, 13, 14, 15, 19, 22) have shown, after the application of growth factors, an increase in cellular activity and, therefore, in regenerative capacity. As mentioned, these factors are found naturally in the body and, when a tissue injury occurs, they are concentrated in the focus of the lesion to promote repair. These studies have shown that the expression of the growth factors and their receptors is modulated after an injury and the cells of the tissues in the process of regeneration are sensitive to the interaction with the growth factors; They have also shown significant increases in cell activity indicator parameters, as well as improved regeneration, through the application, in vivo and in vitro , of various growth factors.

Of the growth factors that are concentrated naturally in the focus of an injury, an important contribution It is released by platelets. Platelets are elements small cell phones with a discoid shape, 2 to 4 micrometers in diameter, heterogeneous in size and density. They are cytoplasmic fragments of megakaryocyte, giant cell of the hematopoietic series found in the bone marrow and it fragments to give rise to platelets in the bone marrow itself or shortly after entering the blood, especially when passing through the pulmonary capillary territory Platelets have many of the functional characteristics of whole cells, although not can reproduce, because they lack a nucleus, which is why neither can they synthesize RNA messengers from which synthesize proteins Some circulate in the blood 8-10 days, at the end of which your cycle ends vital, being eliminated mainly by the macrophage system tissue. Its normal blood concentration ranges between 150,000 and 300,000 per microliter.

Platelets have an important function physiological mechanism of hemostasis, as they contain Several procoagulant factors. Together with the fibrin from of the fibrinogen present in the plasma, the membranes of the activated platelets constitute an important autologous biomaterial related to clot formation. The cell membrane of the platelet contains on its surface a cover of glycoproteins which prevents its adherence to the normal endothelium and causes it to adhere to injured areas of the vascular wall, especially at injured endothelial cells, in even more to any collagen exposed in the depth of the vascular wall. In said membrane large amounts of phospholipids are also found that they play several activating roles in multiple points of the blood clotting process In the cytoplasm, on the other part, there are some structures, called granules platelets, inside which a very complex mixture is stored of proteins, some from the precursor cell (the megakaryocyte) and others captured by torrent endocytosis blood Of the three main types of granules that are found in the cytoplasm of platelets (dense granules, lysosomal granules and α-granules), the most important are the α granules, in which a multitude of substances with diverse functions: adhesive proteins, factors of coagulation, proteases and antiproteases, membrane glycoproteins, basic proteins, bactericidal proteins, cytokines and chemokines, growth factors, fibrinolytic factors and associated factors (such as plasminogen, u-PA, α2-antiplasmin, the glycoprotein rich in histidine, TAF1, α2-macroglobulin or the plasminogen activator inhibitor (PAI-1), that besides having an important function in the regulation fibrinolytic is also able to induce vitronectin so that cell-matrix interactions are formed) and others proteins such as immunoglobulins, albumin or Chondroitin-4-sulfate.

With all this, platelets not only have a important physiological function in the mechanism of homeostasis, thanks to the different procoagulant and promoter factors of the platelet aggregation that it possesses but also an important role in tissue repair and regeneration, thanks in large part to the cytokines, chemokines and growth factors that they transport and they are able to release in places where there are tissue damage All these factors stimulate to a greater or lesser degree Chemotaxis, proliferation and cell maturation. Too are potent angiogenic factors and cell mitogens endothelial (1, 10, 15, 20). In addition, they have the potential to interact therapeutically in those processes in which they are naturally wrapped.

For platelets to release all these Factors must first be activated. When activated, a signaling cascade that leads to the reorganization of the platelet cytoskeleton, granule centralization secretors and exocytosis of small molecules and proteins from the three types of granules: dense granules, granules α and lysosomes.

Among the substances released, the factors of growth released by platelets (which, in most of the cases can also be synthesized by other types cell phones), which are directly involved in the Tissue repair are:

- PDGF (growth factor derived from platelets), which seems to be the first growth factor present in injured tissues, the repair process of the connective tissues and has been identified in fracture foci and in soft tissue wounds in both man and models animals.

- b-FGF or FGF-2 (basic fibroblast growth factor), which, as FGF-1, promotes the growth and differentiation of large variety of cells, including epithelial cells, myocytes, Tenocytes, chondrocytes and osteoblasts.

- VEGF (vascular growth factor endothelial), whose main function is the stimulation of angiogenesis in the areas of injury.

- EGF (epidermal growth factor), which stimulates cell migration towards the focus of the lesion and the mitogenesis

- IGF-1 (growth factor similar to insulin type 1), whose role in repair and remodeling of injured tissues is beginning to be studied and which seems to have an important role in migration and proliferation of the smooth muscle cells (SMC) of the vessels.

- TGF-? (Factor of transforming growth type?), denomination that applies to a superfamily of growth and differentiation factors that It is found in bone, cartilage and platelets, in which it seems synthesized once the clot formed at the site of the lesion, being released by platelet degranulation and affecting the activity of fibroblasts, spinal stem cells and preosteoblates The most important function of TGF-? (16) is chemotaxis and mitogenesis of precursor cells, especially of the osteoblastic line, with the resulting bone formation, and also stimulate the deposit of the collagen matrix in injured connective tissues. Of the family members, are TGF-? 1 and TGF-? 2 those that are most involved in the regeneration of connective tissue and bone repair; he TGF-? 3, on the other hand, has been linked to a anti-catricial activity

The observation that, when a tissue injury, growth factors are concentrated at the level of the focus to favor repair, has led to started to be used in clinic as process enhancers Natural repair of biological tissues by applying them in place of the lesion, thereby increasing its concentration in that area. In the field of traumatology in particular has been shown that growth factors have an important function in the bone and cartilage formation, fracture repair and others injured musculoskeletal tissues, such as tendon lesions (10,13,20).

One of the possible ways of applying these growth factors is the use of the so-called platelet rich plasma (PRP), a term coined to describe a platelet-enriched plasma with respect to the normal concentration that would be found in vivo . According to the criteria of hematologists, a PRP is a plasma that contains over 300,000-350,000 platelets per microliter (2). The objective of its use is to improve surgical evolution, strengthening and enhancing the physiological repair process, as well as allowing a faster and higher quality regeneration in damaged connective tissues. There is a preference for the use of autologous plasma, to avoid adverse reactions of rejection.

Initially, platelet concentrates were they prepared using plasmapheresis systems, a method, commonly used for blood donation, in which by a special machine, with a sterile single-use device and with a just prick, the plasma and the plasma automatically separate platelets and returned to the donor, through the same tube, the rest of blood components (red blood cells and leukocytes). Subsequently different procurement systems have been introduced to the market and preparation of platelet concentrates for therapeutic purposes. Recently, even high-tech equipment has been developed which by centrifugation obtain a platelet rich plasma optimal with less blood drawn (10-50 ml) Preparation protocols vary from one system to another, as well as the volume of blood processed, the volume of product, the platelet concentration obtained, recovery performance of platelets, contacts with sterile barriers, concentration specific to each growth factor and other proteins present, blood processing time, etc. For your comparison (12), the systems are divided into manual and automatic. Among the most prominent are:

1. PVP (double spin)

2. Landsberg (double spin)

3. Clinaseal (single spin)

4. Harvest smart prep . Symphony (automated)

5. Anitua (double spin).

6. GPS (automated)

7. PCCS (automated).

Next, Table 1 shows the characteristics of some of these platelet concentrate processing systems, (extracted from RE Marx et al . (12)):

TABLE 1 Characteristics of different systems for obtaining platelet concentrates

Volume Weather from Volume Concentration PDGF-AB TGF-? 1 from blood indicted from from platelets indicted product Anitua 54 12 9.5 ± 0.5 433 ± 129 35 ± 11.3 52 ± 76 (ml) (min) (ml) (x 10 3 / \ mul) (ng / ml) (ng / ml) Clinaseal 27 17 7.6 ± 0.5 401 ± 267 46 ± 15.3 55 ± 18.7 (ml) (min) (ml) (x 10 3 / \ mul) (ng / ml) (ng / ml) PVP. 43 30 7.8 ± 0.5 493 ± 245 35 ± 17.2 43 ± 17.9 (ml) (min) (ml) (x 10 3 / \ mul) (ng / ml) (ng / ml) Landsberg 52 30 10.6 ± 0.5 433 ± 129 26 ± 13.7 50 ± 10.8 (ml) (min) (ml) (x 10 3 / \ mul) (ng / ml) (ng / ml) Symphony fifty fifteen 11.3 ± 0.5 783 ± 544 97 ± 22.9 130 ± 37.2 (ml) (min) (ml) (x 10 3 / \ mul) (ng / ml) (ng / ml)

When a single stage of centrifugation, starting with whole blood, the different blood components, due to their different density, are located in different fractions along the tube in which they have been subjected to centrifugation, which are distinguishable by the naked eye: lower fraction, red, is the one that corresponds to the red blood cells; immediately above this lower fraction a thin fraction in which the leukocytes are found; above of the leukocyte fraction there is another fraction, lacking leukocytes, in which growth factors are found and platelets, the latter in a concentration that is approximately three times higher than present in the blood peripheral, so it is the fraction that you want to isolate for get platelet rich plasma; the upper fraction, for Finally, it corresponds to the plasma, which interests discarding. Thus, when you want to get platelet rich plasma, the usual thing is discard the upper fraction after having been extracted with a pipette and then extract the fraction immediately located by below which is what, as mentioned, corresponds to plasma rich in factors, without leukocyte contamination and with a platelet concentration approximately three times higher than present in peripheral blood. That fraction can be considered platelet rich plasma (PRP).

Whichever system is used, basically the goal is to get a PRP in the shortest time possible, with a platelet count greater than 300,000 platelets / \ mul. It has been checked with optical microscopy, electronically and with monoclonal antibody techniques, in studies in which these types of procedures have been performed to obtain platelet-rich compounds (2), which platelets do not suffer structural or morphological changes, therefore, are not activated during handling and the content of its α granules remains intact

Prior to the application of PRP in the focus Injury, it will be necessary to induce exocytosis of substances present in the different types of granules: it is what is known as platelet activation. Activation is the beginning of a signaling cascade that leads to the reorganization of the platelet cytoskeleton, the centralization of its granules secretors and exocytosis of small molecules and proteins from the three types of granules: dense granules, granules α and lysosomes. Activation can be achieved, for example, adding calcium chloride to the PRP sample to be used. The optimum calcium chloride concentration should be determined for activate the granular secretion process because it has been proven that, when calcium chloride concentration is excessively increased, there is a decrease in exocytosis, probably due to the protease activation present in platelets (2).

The first clinical applications of PRP they were in oral and maxillofacial surgery (11, 19). The benefit clinical obtained in this specialty is due to the ability to these proteins to accelerate soft tissue repair, as well as bone regeneration in the preparation of future areas for the placement of dental implants.

In 1995 Slater et al . (17) showed that there was a marked stimulating effect on proliferative activity and on the continuity of differentiated activities, including matrix formation and deposition of minerals by osteoblasts in platelet supplemented cultures and that this provided evidence in vitro that support the theory that substances released by platelets could play an important role in fracture repair and could have potential clinical applications in the fracture healing process. In addition, this effect is not limited to osteoblasts.

In this period of the early 90s, we began studying the obtaining of autologous platelet concentrates (platelet rich plasma) and the study of the growth factors contained in them. Initially, he focused on the field of maxillofacial surgery, using platelet gels in the bone graft for mandibular reconstitution, surgical repair of the alveoli and osseointegration of the implants (Whitman et al . (21)). Subsequently, the works of Nash et al . (14) regarding the utility of PDGF in bone consolidation after having performed tibial osteotomies in rabbits, they showed an increase in the density and volume of bone callus in the group treated with PDGF and suggested that the PDGF had a beneficial effect on fractures, They opened the field for the use of platelet-rich plasma in the field of traumatology.

Subsequently, the use of plasma has been tested rich in platelet factors combined with bone graft for cases of pseudoarthrosis, to perform vertebral fusions with autograft and prosthetic implantation.

- Robert E. Marx et al . (11) found that platelet rich plasma increased platelet concentration in the bone graft. They identified the PDGF, TGF-? 1 and TGF-? 2. They indicated that spinal undifferentiated cells had receptors for these growth factors. They obtained platelet factors by density gradient centrifugation. This technique produced a platelet concentration of 338%. Evaluations using monoclonal antibodies showed that spinal cells had receptors for these factors and were able to respond.

In his study on reconstructions mandibular obtained a bone maturation, evaluated by study radiographic, from 1.62 to 2.16 times faster than in those patients treated with bone graft, but without factors of increase.

In Spain, the works of E. Anitua, M. stand out. Sánchez and collaborators (1, 2, 15) pioneers in the study of Platelet rich preparations. They described the formation of a platelet concentrate rich in growth factors, with platelet and plasma proteins, which does not contain leukocytes (one). They conducted many studies on these preparations, and They designed systems to obtain it.

The works of E. Anitua have focused on All fields of maxillofacial surgery and implantology. M. Sánchez has applied this knowledge in orthopedic surgery and traumatology, always focusing on bone regeneration.

Hayneworth et al . (8) studied the action of platelet-rich plasma on mesenchymal stem cells, determining that migration of these cells to the surgical site is stimulated, followed by substantial replication to form a repair blastema. As bioactive growth factors diffuse outward from the fibrin matrix, now densely populated by mesenchymal stem cells, the cells stop dividing and are prepared to respond to residual platelet-rich plasma and endogenous inductive factors that stimulate cell differentiation process.

The use of PRP in the repair of injuries of tendons, however, had not yet been described.

Tendons are structures formed by a regular and dense connective tissue, arranged in beams or fascicles, which attach the muscle to the bone. They are structures specialized in the absorption and transmission of the forces of high tension from the muscle to the corresponding bone, being its main quality the tensile strength, which explains the existence in some cases of tear-fractures. The muscles to be able perform your action and be able to move the joints must be joined by its ends to the bony levers that they will displace.

The main constituent of the tendon is the collagen (86% type I and 5% type III, depending on dry weight) (18). These collagen fibers predominate over elastic ones, responsible for the flexibility and distention of the tendons. The collagen molecules are made up of three chains and it He organizes in microfibrils and finally with his meeting in fibrils. Proteoglycans and glycoproteins along with water are combine with the fibrils of the matrix forming fascicles. The Fascicles merge and give rise to the beams, which are surrounded for the endotenon. The endotenon represents the support of the blood and lymphatic vascularization, as well as innervation. He epitenon and then paratenon constitute structures connective-vascular constitute groups of beams that complete the anatomical structure of the tendon surrounding it.

The union of the tendon in the bone is called direct insertion, depending on the composition and organization of its layers At the ends of the muscles the connective tissue muscle (endotenon, epitenon, paratenon) becomes very fibrous and thick as the number of collagen fibers increases, these form you make parallel to each other that finally get into the matrix calcified collagen, bonding tightly. These fibers of collagen that is introduced into the bone is called fibers from Sharpey.

Tendons are relatively hypocellular structures, fibroblasts line the subunits of fibrils. Proteoglycans are extremely hydrophilic and influence the viscosity and elasticity properties of tendons. Decorin is widely distributed and is considered to play a fundamental role in regulating the formation of collagen fiber formation in vivo (18). It has been assumed that decorin prevents tearing during tendon deformation, thereby increasing its resistance. Compared to ligaments, the deformation induced by the application of a load is less. Therefore, tendons can better transmit the force from the muscle to the bone, allowing movement during contraction or preventing it during eccentric contraction. Characteristically, the tendons transmit the tensile forces, but compression forces occur in the tendons surrounding the articular surfaces. The tendons existing in these areas have a more cartilaginous structure.

The normal tendon is extraordinarily tough and strong. McMaster research is often cited, published in The Journal of Bone and Joint Surgery in 1933. In its experiments, the loads applied to the Achilles tendons of normal rabbits produced joint injuries muscle tendon or bone insertion, but not in the middle of the parenchyma of the tendon. In such preparation there were that section 50% of the tendon fibers so that the tendon give in.

Tendons can be classified in two main categories:

one.

- With Paratendón : they are those that contract in a straight line, without bends or flexions, do not have sheaths but are surrounded by the paratendón, a loose connective tissue that continues with the tendon. This connective tissue develops fibrous adhesions with nearby structures, which prevents the separation of the ends in case of tendon rupture.

2.

- With Mesotendon : those whose contraction is performed in a curve, are surrounded by a tendon sheath that directs the tendon and acts as a pulley. This tendon sheath has two leaves: One visceral, in direct contact with the tendon. Another parietal, away from the tendon. Between both sheets there is a virtual space filled with synovial fluid. The movement of this type of tendon is facilitated by the synovial fluid produced by the synovial membrane or by the epitenon.

The vascular supply of both types of tendon is very different (18). The tendons with sheath receive their vascularization of the perimysium, of the periodic insertion and of branches long and short from the proximal mesotendon. In the tendons surrounded by the paratendón, the vascular contribution comes from capillaries Longitudinal of the periphery. These tendons also receive vascularization of the perimysium and its bone inserts.

The structural and mechanical properties of tendons are key to its force generating function of tension. The force-elongation curve represents the structural properties of the unit bone-tendon-muscle, represented similar to that of the ligaments (18). The properties tendon mechanics depend on the structure of the fibrils of collagen The force-elongation curve indicates the existence of an initial region in which the tendon shows a initial strain without the application of a significant force secondary to the straightening of irregular fibrils and the orientation of longitudinal collagen fibers. This region is smaller in the tendon than in the ligament because the fibers of collagen have a more parallel orientation and therefore require a minor realignment during the initial phase of the contraction. Behind it there is a more linear region that represents the elastic modulus of the tendon. Tendon failure can occur suddenly or gradually. The gradual failure curve, which represents the elastic modulus of the tendon, represents the appearance of permanent structural alterations or elongation irreversible tendon. Young animals with immaturity skeletal show a more sudden curve failure force-elongation than animals with maturity skeletal The viscous and elastic properties of tendons They influence their function. In isometric contractions, the muscle-tendon unit length remains constant, but tendon lengthening occurs due to drag, which results in muscle shortening. This mechanism improves muscle function during isometric contractions. He pre-conditioning takes place when the signs initials of lengthening after a rest period reveal a Increase in energy loss. Behind the pre-conditioning cycle force-elongation can be repeated. This is the reason that warm-up before exercise is key to the proper operation of the unit muscle-tendon-bone.

The anatomical location of the tendons It influences its mechanical and structural properties. In studies differences in biomechanics and biochemistry have been observed significant between flexor and extensor tendons. In the hog the force until the failure in the digital flexor tendons is double than in digital extensor tendons. Also, in the curves force-elongation, hysteresis (loss of strength) is twice as large in tendons as in flexors. The age and maturation increase the difference between both types of tendon.

It has been shown that physical exercise causes positive effects on mechanical and structural properties of the tendons. The elastic module and the force until maximum failure in the digital extensor tendons of the pig increase with the exercise. As demonstrated in biochemical studies, the collagen synthesis and the diameter of the fibrils increase after exercise. The larger diameter fibrils show an increase in resistance properties due to its greater number of links crossed.

In a study on patellar tendons partially or totally protected from stress, those totally protected showed significantly lower tensile strength when studied after one, two, three, six weeks, compared to partially protected tendons. In a study in vitro on digital flexor tendons of the dog totally protected from stress, a significant decrease in the elastic modulus was observed after a period of eight weeks.

The dog's digital flexor tendons showed a significant increase in resistance properties when subjected to cyclic forces, compared to in vitro tendons not subjected to these forces. It has been shown that physical exercise exerts a different effect on different tendons, suggesting that some of the tendons may improve their structural properties while others work close to their maximum capacity. Tendon immobilization decreases its tensile strength, stiffness and total weight due to the reduction in cellularity, in the organization of collagen, in the diameter of collagen fibrils and in the cross-links of collagen, as well as the alteration in its content in proteoglycans and water. The return to mobility results in a slow return to the normality of the biochemical and biomechanical properties of the tendon. This process requires much more time than immobilization and indicates the need for early tendon mobilization whenever possible.

Aging is accompanied by a decrease in the vascularization and cellularity of the tendons. Also gives leading to an increase in the amount of insoluble collagen, of the maturation of collagen crosslinks and the diameter of collagen fibrils, as well as a reduction in turnover metabolic of collagen, and of the content of proteoglycans and of Water. These modifications could be evident during the third decade of life and make the tendon weaker, more rigid and less distensible Additional degenerative changes in the tendon increase the chance of injury. Physical exercise It can be useful to delay these changes associated with age.

The incipient changes are characterized by granularity and loss of the normal wave contour collagen fibers and the bundles thereof. The structures They assume a fairly homogeneous image. Tissue cells connective are distorted and the parallelism of the fibers The cell nucleus is distorted, that is, some are rounded, others are picnotic or fasciculated. Some areas of the tendon have a gelatinous or edematous appearance, with laxity of the fibers, which contain broken elements frayed, separated by a homogeneous material that almost does not capture the dye

Musculoskeletal lesions They are very common. In series of athletes its incidence varies from 10% to 50% of all injuries (18).

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A picture of tendon degeneration that is acquiring great relevance for its prevalence is that of the cuff of shoulder rotators. Given the anatomical conditions and exposed biomechanics, this tendon structure is vulnerable in individuals subjected to repeated efforts, especially in those they present unfavorable anatomical forms for the correct shoulder mechanics If the alterations that occur last during time, they are extended to neighboring structures and compromise, so that they end up participating in the consequences of the injury that started in the cuff, leading to to more complex clinical pictures. In increasing lesion severity We found: supraspinatus tendonitis, tendinitis with calcification, incomplete rupture and complete rupture, as picture More serious.

Tendon lesions are frequent and are responsible for substantial morbidity in both sport and in everyday life (18). They take place through three mechanisms. Direct trauma can result in the tendon section. Indirect injuries consist of tearing the tendon since your bone insertion. The indirect intrathendinous lesion may be due to intrinsic or extrinsic factors (such as lacerations of the tendons of the hand). Excessive forces applied on the bone-tendon unit can give place to bone removal (as occurs in lesions due to tearing of the deep digital flexor of the ring finger). The repeated submaximal overload or repetitive pressure against a bone surface, as seen in the supraspinatus under the acromion, can lead to tendon degeneration.

Rat Achilles tendons have been used and of the rabbit to study tendino extrasinovial healing (18).

The healing of the tendon after an injury is similar to the wound repair response seen in other soft tissues The three phases of healing take place (inflammatory, fibroblastic and remodeling):

Inflammatory phase :

After tendon injury, the solution of continuity is filled with inflammatory products that come from adjacent tissues, such as blood cells, remains Nuclear and fibrin. Inflammatory cells phagocytize tissues Necrotic and clot.

Fibroblastic phase :

Fibroblasts and hair buds too occupy the continuity solution between the ends of the tendon, and proliferate from paratenon. After three days you can demonstrate the synthesis of collagen and other matrix components extracellular Type I collagen production increases by 15-22 times.

After two weeks a bridge is observed fibrous consisting of fibroblasts and collagen fibers that bind The tendon

Remodeling phase :

After three to four weeks, the fibers of collagen begin to organize longitudinally along the tendon in a process that continues for many months.

Fibroblasts are the main cells in fibrotic repair, being responsible for the deposit of collagen and scar formation.

During the early stages of healing, the scar is much larger than the tendon injury, but gradually decreases in size during healing due to partial reorganization of collagen and link establishment crossed between collagen fibrils. This process improves with the proper application of forces.

Occasionally, the production and deposit of Collagen fibers is excessive. This exaggerated collagen deposit it distorts the architecture and, with it, the function of the organ in the that occurs, giving rise to the so-called "fibrosis".

Two tendon repair mechanisms have been classically proposed:

1. Extrinsic mechanism :

In which it is proposed that fibroblasts and inflammatory cells of the periphery invade the lesion focus and promote tendon repair.

2. Intrinsic mechanism :

In which the proposal is that fibroblasts and the inflammatory cells come from the tendon itself and epitenon

The healing process in surrounded tendons per pod has been the subject of investigation for many years, observed in the clinic that the mechanisms are combined intrinsic and extrinsic.

The differences between the intrinsic mechanism and extrinsic extend beyond the origin of the cells reparative The extrinsic mechanism appears at an early stage, while the intrinsic mechanism appears in one more phase belated

In the first studies it was pointed out that the healing took place by forming a great response inflammatory and proliferative from the synovial sheath, forming a granulation tissue.

In more recent studies it has been shown that tendon cells participate in the healing response (18).

However the inflammatory response and proliferative that occurs from the synovial sheath is much more extensive compared to tendon and epitenon response (9).

It has been observed that in epitenon cells and mRNA endotenon of the flexor tendon in healing phase of pro-alpha-1 collagen.

In the flexor tendons in healing phase in those who develop passive controlled movements, the Intrinsic healing response of epitenon is the way Healing predominantly. In tendons with immobilization, the Main healing response is due to granulation tissue coming from the sheath and cell proliferation in the endotenon. During the intrinsic healing response, synthesis of collagen and vascular response followed by a maturation of collagen and a reorganization similar to those that take place in the repair responses seen in other tissues.

Other studies have shown that fibroblasts synovial are more reactive to cytokines and have great ability to degrade the extracellular matrix (9, 18). It has also thought that the predominance of the extrinsic mechanism increases the collagen content in the lesion focus, as well as reduces the level of collagen organization and tissue properties tendon repair. For this reason, it is thought that the predominance of the extrinsic mechanism leads to the formation of scars and adhesions between the tendon and structures peritendinous.

Trying to favor the intrinsic mechanism (increasing terminal-terminal healing), while an attempt is made to suppress the extrinsic mechanism (reducing the tendon adhesions with peripheral tissues) could improve the treatment of these injuries, because the decrease of the mechanism extrinsic in favor of the intrinsic mechanism would decrease the probability of a messy blockbuster of collagen and, with it, the probability that produce fibrosis

The main factors that influence the tendon repair are:

- The type of suture repair.

- Passive mobility continues.

- The formation of continuity solution.

- The force experienced by the tendon in the healing phase

It has been shown that movement and load application, early and actively increase the incidence of tendon rupture and solution formation of continuity.

On the other hand, it has also been shown that early and controlled passive mobilization improves various aspects of tendon repair. Passive and controlled mobility improves the strength of the reparative tissue during the initial period of the repair, and decreases adhesions.

Continuity Solution Formation (elongation) in the repair area of the flexor tendons in Healing phase has been associated with adhesion formation and poor functional results.

In a study in which the continuity solution formation in the canine flexor tendon in healing phase has shown a significant increase in the Ultimately existing force, in the stiffness of the area of retraction and stretching of the repair area in tendons with a continuity solution less than 3 mm, compared to tendons with a continuity solution greater than 3 mm. These differences were observed at 10 and 42 days by mechanical tests, and no differences were observed in the prevalence of adhesions (7).

There is controversy in the relationship between the type of suture and the application or not of rehabilitation. Thus Boyer et al . (4), in their study with canine flexor tendons they concluded that vigorous rehabilitation could help improve function, however they found no evidence that it enhances tissue repair or strength in the context of new sutures.

The objective of surgical treatment in tendon repair is to make a primary repair with sufficient tensile strength to allow rehabilitation through passive movements that prevents the formation of adhesions and facilitates repair (5). However, like any other cut that occurs in the body, surgical treatments involve subsequent healing, which involves the repair of tissue that was cut with fibrous tissue. It is not uncommon, however, that the scarring is excessive, so that more fibrous tissue is formed than is necessary. When this excessive accumulation occurs during the healing following a surgical process, it is called post-surgical fibrosis, a term that is generally used to indicate that excessive fibrous tissue formation has occurred during the healing process secondary to the surgical operation and not to simply refer to scarring secondary to any surgical operation, as could be understood if used in its strictest sense. As a consequence of this process, the injured tissue will be transformed into fibrotic tissue, altering the normal characteristics of the tissue. Thus, for example, in the case of epidural fibrosis, which occurs in most of the operated vertebral columns, especially when they are operated to solve the cases of displacement of the intervertebral discs known as herniated discs, a replacement of normal epidural fat by postoperative fibrotic tissue. If, after a spinal operation, that excessive fibrotic tissue compresses a nerve, it causes large
pains

There are also other cases of fibrosis that do not They are secondary to surgical processes. This is the case, for example, of cystic fibrosis (CF), one of the genetic diseases most common hereditary in the white race. In patients who they suffer from it, the body produces an excessively viscous mucus that plug the airways, making breathing difficult and causing serious infections. It also blocks the ducts of the pancreas, making food assimilation difficult, causing in patients malnutrition an abnormal development. The illness, autosomal recessive, shown from the moment of birth, by what is considered important early diagnosis and administration of adequate treatment to prevent it from reaching severe deterioration of lung tissue, which can reach to make a transplant necessary.

Fibrosis is also usually of genetic origin progressive palmar fascia, also known as disease of Dupuytren, in which the fibrotic tissue wraps the tendons flexors, particularly those of the fourth and fifth fingers. This produces finger and palm contracture, but may need decades to develop Although its main component is genetic, It also looks like post-traumatic scarring or in chronic liver disease. Repair of the flexor tendons of the hand is one of the aspects in which the usefulness of knowledge related to the biology of growth factors (3).

Fibrosis is also one of the components fundamentals of chronic liver damage, whose final phase is the cirrhosis. During its development, a process of fibrogenesis that leads to the abnormal accumulation of collagen and that results from the alteration of the homeostatic mechanism between different cell lines of the liver and of them with the matrix extracellular An anatomical alteration occurs at the cellular level of the structure of the liver, decreasing its functionality.

Muscle fibrosis is also common, in the that muscle fibers lose the ability to contract and They transform into fibrous tissue. With this, the function disappears contractile and, in addition, it is painful.

Given the high frequency with which they occur tendon lesions, it would be interesting to have compositions that allowed to reduce the development of fibrosis during the healing process of these injuries and, in general, during all tissue repair processes that occur as a result of cuts in said tissues, physiological processes in which there is a risk of development of secondary fibrosis to them. It would also be interesting to arrange of compositions that would limit the development of fibrosis in patients suffering from diseases that normally occur with the development of fibrotic processes that are the main cause of symptoms of the disease or that aggravate the symptoms of same, diseases that include cystic fibrosis and chronic liver damage The present invention describes that plasma platelet rich may be useful to limit development of fibrosis, whether it is likely to occur in a tissue repair process as if its development is characteristic in the course of diseases in which fibrosis produced is the main cause of the symptoms of the disease or aggravate the symptomatology of the same, so the use of plasma rich in platelets to prepare medicines to combat fibrosis, whether administered preventively, to avoid or control its development, as if they are administered for therapeutic purposes, to reduce the severity of states fibrotic already established.

Description of the invention

The invention relates to the use of rich plasma in platelets (PRP) for the preparation of a medicine for Fight fibrosis

As used herein, the term "combat" has the meaning of "attack" what that is considered a damage, both with the objective of opposing its development trying to limit or prevent it, as with the objective of mitigating its severity once it has occurred. It is because of that The medicaments referred to in the invention may be intended to be administered for both preventive purposes, it is say, be administered before or during the course of a process in which may occur fibrosis, to prevent it from occurring or decrease its development, or they may also be intended to be administered to mitigate the severity of a fibrosis already established.

In the event that the medication is planned to be administered before or during the course of a process in the that fibrosis development may occur, the process during which fibrosis may appear may be a process of tissue repair In a preferred embodiment of this aspect of the invention, the tissue repair process in which you want combating the development of fibrosis is the repair of lesions of tendons The injured tendon can be any tendon, although he especially prefers that the medicine prepared from Platelet-rich plasma is applied when the tendon to be repaired is the Achilles tendon, a flexor tendon or a hard-to-reach tendon surgical, such as the tendons that form the cuff of the rotators, especially the supraspinatus tendon. The injury in the tendon may have assumed that the tendon has been completely sectioned, as in the example shown below herein, or a partial, less complex injury, in the that the tendon has been injured but in which it is not it is necessary to completely religionize two tendinous ends that have It was completely separated. Since in the breaks tendons, as shown later in an example of regeneration of sectioned Achilles tendons, the use of Platelet-rich plasma promotes fiber regeneration tendons, decreasing the occurrence of fibrosis with respect to spontaneous repair processes in which plasma was administered rich in platelets, in less complex lesions plasma rich in Platelets must also be effective.

In another embodiment of the invention, the process of tissue repair in which you want to limit the development of Fibrosis is a tissue repair process secondary to a process surgical. In that case, the medicament of the invention will be intended to be applied in a place of the body that has been subject to a surgical process, in order to avoid the development of fibrosis during the tissue repair process that takes place after the surgical process. The surgical process, for example, may have been performed to facilitate the repair of the lesion of a tendon, a tendon that can be, for example, an Achilles tendon, a flexor tendon or one of the tendons that form the cuff of the rotators, especially the supraspinatus tendon, the latter tendon which is subject, with relative frequency, of bursectomy, acromioplasty and repair (suture-reanimation of same). Another tissue repair process secondary to a process surgical in which the use of medicines that contain platelet rich plasma are the spinal operations, especially those intended for treatment of herniated discs, in which it could be applied to limit the appearance of excess fibrotic tissue during the post-surgical process, avoiding or decreasing the magnitude of the complications that excessive fibrotic tissue can cause such as compression of a nerve, causing the alteration of its normal functioning and / or causing pain.

Another of the possible applications of medicines prepared from platelet rich plasma is limit the development of fibrosis in processes that do not constitute strictly tissue repair processes, but they are the process of evolution of a disease that normally occurs with the fibrosis development One of the examples of these diseases is cystic fibrosis, in which the fibrosis produced during evolution of the disease is blocking the airways, making breathing difficult, and also blocks the ducts of the pancreas. An early diagnosis is considered essential so that the appropriate treatments are developed as soon as possible and, among the treatments to be administered could be medications prepared from platelet rich plasma, to help limit the development of fibrosis and prevent it from reaching severe deterioration of lung tissue.

Another disease that normally occurs with the development of fibrosis is chronic liver damage, which in Its final phase results in the onset of cirrhosis. The administration of drugs prepared from platelet rich plasma could also help limit the development of fibrosis during the disease process, mitigating the severity of the disease.

It is also an example of disease normally with the development of fibrosis the so-called disease of Dupuytren or progressive fibrosis of the palmar fascia, in which the fibrosis wraps the flexor tendons, particularly those of the fourth and fifth fingers It is a disease that may need decades to develop The administration of a medicine prepared to starting platelet rich plasma could also help limit the development of fibrosis, lengthening the time needed for a complete development of the disease or even, preventing that the contracture of the fingers and the palm that occurs It occurs in advanced phases of it.

The invention also contemplates that medicines prepared from platelet rich plasma are design to be used to remit the severity of states fibrotic already established. These fibrotic states may have produced in the course of the evolution of diseases of the type before mentioned, such as cystic fibrosis, chronic liver damage or Dupuytren's disease, or they may have occurred during a tissue regeneration process that may or may not be secondary to a surgical intervention.

In the formulation of prepared medicines from platelet-rich plasma intended to combat fibrosis may include any suitable excipient pharmaceutically acceptable, as well as, optionally, others active ingredients, which may also be intended to combat fibrosis, or may, for example, be intended to treat others effects linked to the regenerative process on which it is desired to act or to the disease that normally occurs with associated fibrosis.

The composition of the drug and the mode of administration for which it is planned will vary depending on the fabric in who wishes to limit the development or decrease the severity of the fibrosis and the process or disease on which you want to act. In the case of fabrics easily accessible from the outside, as is the case of Achilles tendons or flexor tendons, you can use subdermal injection in the lesion focus; this same method of administration is valid for external scars since produced as pronounced grooves or scar depressions. In those cases, the medicine may also contain fat in your composition and be prepared to be administered as a graft. Another possibility in the case of easily accessible fabrics from the Outside is the use of mesotherapy, a technique that involves intradermal infiltration of the composition to be administered to a subject by Assisted Percutaneous Route, which represents the Penetration of drugs through the skin by:

?

sonophoresis: introduction of substances inside the body through energy ultrasonic

?

electrophoresis: the charge electric makes the molecules move

?

iontophoresis: application of ions and ionized molecules in the skin by current galvanic

?

hydroelectrophoresis: through pulsed currents

?

cryoelectrophoresis: current pulsed electric

Subdermal injection and mesotherapy are especially interesting when it comes to difficult tendons surgical access such as those that, on the shoulder, are part of the called rotator cuff, especially the tendon supraspinatus, tendon in which they often occur painful injuries in the process of repair, or after same, a drug prepared from platelet rich plasma to limit the development of fibrosis and help make the final result of the regenerative process better, or also to combat already produced fibrosis. Treatments current more widely spread to promote regeneration of the supraspinatus tendon (rehabilitative treatment together with analgesics and anti-inflammatory in early stages, infiltrations premises with local anesthetics and release corticosteroids delayed for the treatment of severe pain, and treatment surgery consisting of bursectomy, acromioplasty and repair with tendon reassembly and suture in complex cases) are cause to often of undesirable side effects due to or medication provided or to complications of surgical intervention derived from the existing and proper surgery techniques such as infections and bleeding

In the case of regeneration processes of tissues easily accessible from the outside, the medicine can also be in the form of ointment, especially when it comes to A process of skin regeneration.

In the case of hard-to-reach organs, the administration of the medicine prepared from plasma rich in Platelets will be performed by direct perfusion techniques in the damaged organ (helped by simple surgical techniques or by the portal system or by hepatic artery). Both in the case of liver as in the case of pulmonary fibrosis, a guided puncture-injection by CT or ultrasound to reach specific areas of these organs.

As used in the invention, it is called "fibrosis" to an increase in the production and deposition of collagen fibers that are considered exaggerated, for distorting the architecture and / or the normal function of the organ. As used in the invention, a "fibrotic state" is a fibrosis already established, that is, a situation where there is already a deposit of collagen fibers that you may consider exaggerated for being already distorting the architecture and / or normal function of the organ in the one that is located.

As used in the present invention, it is denominate "platelet rich plasma" to any preparation of plasma in which platelets are found at an equal concentration or greater than 300,000 platelets per microliter. Although, as per commented earlier and how can it be deduced from the data of Table 1, the commercial procedures currently available seem to be more effective and lead to less variability in the final concentrations obtained (12), so which are the preferred procedures for obtaining plasma rich in platelets, the invention encompasses the use of plasma rich in platelets obtained by any possible method, both manual and automatic, for the preparation of compositions intended for decrease fibrosis development in regeneration processes tissue.

When using plasma rich in platelet factors or any other type of platelet preparation, a multiple combination of proteins with interactions and effects varied in the different cellular mechanisms (1). It seems like a excessive simplification, for such a complex situation, thinking that the more platelets the better and the greater the clinical effect, or that these preparations only have an effect when they contain concentrations very high platelets per microliter; in fact, there are studies experimental where it has been shown that a concentration effective and sufficient for the desired effect to occur not It involves large doses (2). Therefore, the invention relates to the use of platelet rich plasma for the preparation of compositions aimed at decreasing the development of fibrosis in processes of tissue regeneration regardless of the final concentration of platelets that are finally present in said composition.

Tissue regeneration is a very complex process, as vascularization and cell connections intervene. Therefore, it is convenient to carry out studies in animal models to verify the applicability and usefulness of any product in favoring said process. As described later in the Examples, the comparison of sectioned tendons in which PRP had been injected prior to performing the suture with sectioned tendons in which PRP had not been injected, after three weeks, allows to observe a manifest decrease in the appearance of fibrosis in histological sections corresponding to tendons of animals in which PRP had been injected, without any negative influence being observed on the rest of the parameters observed regarding the evolution process, such as elastic resistance to breakage or cell sorting. Therefore, the use of PRP to prepare medicines intended to be applied in tissue regeneration processes in general and in those of tendon regeneration in particular is shown as a useful means to prevent or decrease the development of fibrosis during said regeneration process. . The use of autologous PRP would also prevent the occurrence of adverse effects due to possible rejection reactions, so one of the embodiments of this invention is that the platelet-rich plasma used is autologous plasma. On the other hand, the concomitant administration of some other agent intended to reduce the appearance of immune rejection reactions would allow the use of compositions prepared from PRP from other individuals, thereby facilitating their large-scale manufacturing and presentation in commercial forms. easy to obtain and handle, so the use of non-autologous plasma for the preparation of the drug is also within the scope of the
invention.

The invention will now be described in more detail. thanks to the Figures and the Example that appear below.

Brief description of the figures

Fig. 1 shows histological sections, stained with Masson's trichrome reagent, from tendon regions of Achilles in which tissue regeneration had occurred that had given rise to their religion after being Tendons sectioned three weeks before.

Fig. 1A shows the staining of a cut histological, which appears blue-green (collagen fibers), corresponding to an Achilles tendon to which no platelet-rich plasma had been applied after sectioning the tendon.

Fig. 1B shows the staining of a cut histological, which appears blue-green (collagen fibers), corresponding to an Achilles tendon to which platelet rich plasma had been applied after sectioning the tendon.

Example

To study the in vivo effect that the use of growth factors could have on tendon regeneration after an injury, animal models were used, rabbits to which the Achilles tendon was sectioned.

Three new white rabbits were used Zealand that were placed in an immobilization cage. After Sedation applying ether, the surgical field was prepared by shaving the here zone in both lower extremities.

Using a percutaneous technique, with a scalpel fine, both Achilles tendons were sectioned in rabbits (tendons which, in the following, are also referred to as "tendons here").

The solution of clinically was checked continuity produced by separating both tendon ends by effect of muscle retraction.

Platelet-rich plasma was obtained in a manner autologous of each rabbit and, after manual processing, was applied about injured tendons. To prepare the plasma rich in platelets, the following steps were followed:

a) Obtaining venous blood: A peripheral route catching a vein on the back of the pavilions Rabbit headphones. 5 cm3 of blood were obtained venous, collected in a tube with sodium citrate as anticoagulant. This anticoagulant was preferred because others induce changes in the platelet morphology or interfere with its aggregation; So, when blood is drawn on EDTA (acid ethylenediaminetetraacetic acid), platelets swell and are shaped spherical instead of being discoid, while another anticoagulant commonly used, ACD (dextrose and citric acid solution), has lower pH and interferes with platelet aggregation (2).

b) Separation of blood fractions by centrifugation It was performed on the tubes used to collect the blood, by a single stage of centrifugation. This was done for 8 minutes at 1800 r.p.m.

c) Isolation of the platelet rich fraction. It was preceded by the extraction of the tube from the upper fraction or plasma, which was discarded. Subsequently, it was extracted with another pipette the fraction located in the cubic centimeter located immediately below the upper fraction that was just discard. This fraction was considered plasma rich in platelets (PRP).

For platelets to release the factors that they contain, they must first be activated. As commented previously, when activated, a signaling cascade starts leads to the reorganization of the platelet cytoskeleton, the centralization of its secretory granules and exocytosis of small molecules and proteins from all three types of granules: dense granules, α granules and lysosomes.

To have a sufficient amount of factors, an easily manageable volume and being able to standardize the technique of obtaining, samples of 1 ml of the fraction were used considered platelet rich plasma (PRP). These samples are activated by the addition of 10% calcium chloride (9.13 mEq / 10 m1), (B. Braun Medical, Rubí, Barcelona, Spain). As it commented earlier, an excessive increase in the concentration of calcium chloride results in a decrease in exocytosis, probably due to the activation of proteases present in the platelets (2), so it is important to determine the amount optimal calcium chloride to add. In this case, it was determined that the optimum amount was 50 µl of 10% calcium chloride per every milliliter of PRP.

Once the platelet rich plasma is obtained activated, the activated sample of one milliliter is applied by percutaneous injection in the lesion focus. In this case, the plasma plate-rich was introduced at the point of section of the tendons Here are the rights of the rabbits. In the hereleal tendons left, which had also been sectioned, PRP was not applied, thus being able to compare the effect of platelet rich plasma on tendon regeneration with respect to a treated control without Plasma in the same model, the same animal.

Once this procedure was completed, the mini-incision using a staple suture, suture which exclusively affected the skin to subcutaneous tissue but never to the structure under study, which in this case were the tendons here Finally the area was cured with povidone iodine solution and an antibiotic prophylaxis was performed.

The staples were removed after 10 days, checking the closure of the incisions.

The evolution of the repair was allowed to follow tendonous for 3 weeks, which is the average healing period of connective tissues. After that period of time, He proceeded to tendon analysis. In this case, the elastic tensile strength and analysis was performed microscopic by histological cuts and stains.

In order to perform the analysis, the Achilles tendons dissected, the area was marked on them where the regeneration was observed macroscopically and performed a first clinical-macroscopic analysis of the tendon regeneration, they were placed in a tensioner calibrated in Kg. Increasing stresses can be applied in this, until causing the tendon rupture, which served to analyze your resistance in a quantitative way.

When applying increasing stresses on the shaft of the tendon, it was possible to check with what tension broke.

In the experimental series a certain increased elastic resistance to breakage of those tendons treated with platelet rich plasma. However, the differences obtained in the study were not statistically significant (possibly due to the small number of animals involved in the experiment).

Once the resistance was evaluated, the tendinous remains of the previously marked area, corresponding to the regenerated region, to be analyzed histologically. After performing histological sections, samples taken from each of the tendons using, on the one hand, the stains of hematoxylin-eosin (staining that results in the cell nuclei appear blue by acid reaction nucleic with the basic hematoxylin dye, while the cytoplasms stain pink or red by protein reaction basic present in it with the eosin acid dye) and, on the other, Masson's trichrome reagent (which stains the fibers red muscle and bluish-green collagen, being specific for see the fibers of the latter).

Histological analysis of the lesion focus showed The following results:

- Staining with hematoxylin-eosin : No significant differences were found, except for a slight increase in nuclei in sections treated with platelet rich plasma; Normal cell sorting in both cuts.

- Masson's trichrome staining : An increase in fibrosis was observed in an unstructured way (motley aspect) in the cuts corresponding to tendons in which platelet rich plasma had not been applied (Fig. 1A) with respect to the cuts in the that platelet rich plasma had been applied (Fig. 1B).

The manifest decrease in the presence of fibrotic tissue in cases treated with platelet rich plasma indicates that this preparation and compositions containing it are useful for use as a limiting agent for the development of tissue fibrosis when this fibrosis is secondary to the process physiological tissue repair, compositions that can be useful also to cause a decrease in fibrosis in Fibrosis tissues already established.

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Claims (29)

1. Use of platelet rich plasma for Preparation of a medicine to fight fibrosis. 2. Use according to claim 1, wherein the medication to fight fibrosis is meant to be administered during a process in which fibrosis can occur to limit its development during it. 3. Use according to claim 2, wherein the medication to fight fibrosis is meant to be administered during a tissue regeneration process to limit the development of fibrosis during it. 4. Use according to claim 3, wherein the tissue regeneration process during which it is desired to limit the Fibrosis development is secondary to a surgical process. 5. Use according to claim 4, wherein the Surgical process has been performed on a tendon. 6. Use according to claim 5, wherein the Surgical process has been performed on a flexor tendon. 7. Use according to claim 5, wherein the surgical process has been performed on a tendon supraspinatus 8. Use according to claim 5, wherein the Surgical process has been performed on an Achilles tendon. 9. Use according to claim 4, wherein the Surgical process has been performed on the spine. 10. Use according to claim 9, wherein the surgical process performed on the spine was going aimed at treating a herniated disc. 11. Use according to claim 3, wherein the tissue regeneration process during which it is desired to limit the Fibrosis development is not secondary to a surgical process. 12. Use according to claim 11, wherein the tissue regeneration process is a regeneration process of a injured tendon 13. Use according to claim 12, wherein the injured tendon has been sectioned. 14. Use according to any of the claims 12 or 13 in which the injured tendon is an Achilles tendon. 15. Use according to any of the claims 12 or 13, in which the injured tendon in a flexor tendon. 16. Use according to any of the claims 12 or 13, in which the injured tendon is a tendon supraspinatus 17. Use according to claim 2, wherein the process in which fibrosis can occur is the process of evolution of a disease that normally develops with development of fibrosis 18. Use according to claim 17, wherein the disease during whose evolution process the medication is intended to be administered is the progressive fibrosis of the fascia palm grove. 19. Use according to claim 17, wherein the disease during whose evolution process the medication is intended to be administered is cystic fibrosis. 20. Use according to claim 17, wherein the disease during whose evolution process the medication is intended to be administered is chronic liver damage. 21. Use according to claim 1, wherein the medication to fight fibrosis is meant to be administered to mitigate the severity of fibrotic states since established. 22. Use according to claim 21, wherein the fibrotic state has occurred during the evolution process of a disease that normally occurs with the development of fibrosis. 23. Use according to claim 22, wherein the disease is the progressive fibrosis of the palmar fascia. 24. Use according to claim 22, wherein the Disease is cystic fibrosis. 25. Use according to claim 22, wherein the Disease is chronic liver damage. 26. Use according to claim 21, wherein the fibrotic state has occurred during a regeneration process tissue. 27. Use according to claim 26, wherein the tissue regeneration process during which the Fibrotic state had been secondary to a surgical process. 28. Use according to claim 26, wherein the tissue regeneration process during which the fibrotic state had not been secondary to a process surgical. 29. Use according to any of the claims precedents, in which platelet rich plasma used to Preparing the medication is autologous.