WO2020016476A1

WO2020016476A1 - Injectable material for joint cartilage regeneration

Info

Publication number: WO2020016476A1
Application number: PCT/ES2019/070505
Authority: WO
Inventors: José Luis GÓMEZ RIBELLES; Gloria GALLEGO FERRER; Carmen ANTOLINOS TURPÍN; María SANCHO-TELLO VALLS; Carmen CARDA BATALLA
Original assignee: Universitat Politècnica De València; Fundación Incliva; Universitat De València; Consorcio Centro de Investigación Biomédica en Red, M.P.
Priority date: 2018-07-19
Filing date: 2019-07-18
Publication date: 2020-01-23
Also published as: ES2690392A1; ES2690392B2

Abstract

The invention relates to an injectable material for joint cartilage regeneration, the implantation of which in the site of a cartilage defect can be combined with a subchondral bone stimulation technique. More particularly, the invention relates to an injectable material containing microspheres of plasma rich in platelets obtained from a blood sample from the patient and other synthetic microspheres, preferably of a bio-reabsorbable polymer, and to its use as a cartilage regeneration support.

Description

INJECTABLE MATERIAL FOR THE REGENERATION OF CARTRIDGE

ARTICULATE

FIELD OF THE TECHNIQUE

The present invention falls within the biomedical and pharmaceutical sector, more specifically, in the field of tissue regeneration, and refers to an injectable material whose implantation in the place of a cartilage defect can be combined with a technique of stimulation of the subchondral bone

STATE OF ART

Currently, articular cartilage pathologies are one of the main causes of disability in the elderly. In the regeneration of articular cartilage, various subchondral bone stimulation techniques are used. These surgical techniques are based on damaging, by different procedures, the bone below the site of damage to the cartilage so that bleeding occurs that opens the way to the migration of pluripotential, mesenchymal cells, with the ability to produce new cartilaginous tissue . However, very often the tissue formed does not have the structure or properties of articular cartilage (hyaline cartilage) but is more like fibrocartilage, softer, which is not functional and degenerates over time. One of the reasons why the cells that reach the defect site are not able to generate the correct tissue is that the biomechanical environment they find is not adequate. Different implants have been developed that are intended to protect the cells and make the dynamic compression loads to which the joint is subjected to transfer to the cells in a similar way as in healthy tissue.

Platelet rich plasma (PRP) is a product currently used in regenerative therapies. It is obtained from an extraction of the patient's own blood, which by centrifugation is separated into different components usually used in the clinic. In the lower part of the centrifuge tube the phase rich in red blood cells is obtained and on it a layer that contains the blood plasma where most of the platelets remain and which has been called in the scientific literature and in the clinical application rich plasma in platelets or PRP. On it are different layers of blood plasma with lower platelet content and is called "platelet poor plasma" or PPP. Both PRP and PPP coagulate in the presence of calcium ions, to form a gel Fortier et al. (The Role of Growth Factors in Cartilage Repair, Clinical Orthopedics and Related Research 2011; 469 (10): 2706-15) and Dold et al. (Platelet-Rich Plasma in the Management of Articular Cartilage Pathology: A Systematic Review, Clinical Journal of Sport Medicine 2014; 24 (1): 31-43), analyze published studies on the application of PRP for the treatment of cartilage pathology articulate.

Some examples of biocompatible materials are disclosed in WO2013116791 A1, which describes a composition comprising a synthetic and biodegradable biomaterial, and a blood extract. The biomaterial can be a polymer and also comprises a hydrogel. The blood extract can be platelet rich plasma. This composition is used in the treatment of skin wounds, osteoarthritis, heart muscle damage, bone defects, traumatic and dental damage. Document W00200272A2 also refers to a polymeric gel composition that, mixed with whole blood, promotes the regeneration of tissues such as cartilage, meniscus, ligament or tendon among others.

In the document by Saito et al. (Intraarticular Administration of Platelet-Rich Plasma with Biodegradable Gelatin Hydrogel Microspheres Prevenís Osteoarthritis Progression in the Rabbit Knee, Clinical and Experimental Rheumatology 2009; 27 (2): 201-7), biodegradable hydrogel materials and intra-articular administration of Biodegradable microspheres of hydrogel-shaped gelatin containing platelet-rich plasma and its effect on the progression of osteoarthritis in rabbit's knee.

W02006023803A2 discloses an implant formed by the combination of microparticles of a polymer as polylactic acid and an autologous component of the patient as blood plasma. This implant can be used to repair articular cartilage by injection. In turn, document W02011150328A1 describes a matrix that promotes cell growth, formed by a biodegradable polymer together with a physiological solution that can be platelet rich plasma.

Various procedures have been published in the scientific literature to produce coagulation of proteins in aqueous media by chemical reactions that include the modification of the protein by grafting in its chain of reactive groups containing carbon-carbon double bonds (C = C) that they are capable of reacting with monomers containing two of these groups C = C, reactions initiated by photosensitive primers activated by visible light or ultraviolet light. These reactions lead to the formation of polymeric networks of the protein, which are capable of absorbing large amounts of water but are insoluble, that is, they form hydrogels (Almany and Seliktar, Biosynthetic Hydrogel Scaffolds Made from Fibrinogen and Polyethylene Glycol for 3D Cell Cultures, Biomaterials 2005; 26 (15): 2467-77; Hu et al. Gelatin Hydrogel Prepared by Photo-initiated Polymerization and Loaded with TGF-bI for Cartilage Tissue Engineering, Macromolecular Bioscience 2009: 9 (12): 1194-201; Oss- Ronen and Seliktar, Polymer-conjugated Albumin and Fibrinogen Composite Hydrogels as Cell Scaffolds Designed for Affinity-based Drug Delivery, Acta Biomaterialia 2011; 7 (1): 163-70; Dikovsky et al. The effect of Structural Alterations of PEG-fibrinogen Hydrogel Hcaffolds on 3-D Cellular Morphology and Cellular Migration, Biomaterials 2006; 27 (8): 1496-506). In the document Pradhan et al. (A Three-Dimensional Spheroidal Cancer Model Based on PEG-fibrinogen Hydrogel Microspheres, Biomaterials 2017; 115: 141-54), it has been shown that, under the appropriate reaction conditions, at a temperature of 37 ° C, with moderate initiator contents and short reaction times, the reaction is compatible with cell viability and therefore allows to obtain hydrogels loaded with viable cells.

In the documents Sakai et al. (An Injectable, In Situ Enzymatically Gellable, Gelatin Derivative for Drug Delivery and Tissue Engineering. Biomaterials 2009; 30 (20): 3371-7), Kuriwawa et al. (Injectable Biodegradable Hydrogels Composed of Hyaluronic Acid-Tyramine Conjugates for Drug Delivery and Tissue Engineering. Chemical Communincations 2005; 14 (34): 4312-4), another way of producing hydrogels of proteins or polysaccharides in the presence of cells is described, maintaining their viability, is the enzymatic crosslinking. Poveda-Reyes et al. (Reinforcing an Injectable Gelatin Hydrogel with PLLA Microfibers: Two Routes for Short Fiber Production, Macromolecular Materials and Engineering 2015; 300 (10): 977-88) modified gelatin by introducing tyramine groups, which dissolved in aqueous medium by suspending mouse fibroblasts in it of line L929. Hydrogel formation was performed by adding horseradish peroxidase (horseradish peroxidase) and hydrogen peroxide, demonstrating gel formation and cell viability after the reaction.

The present invention relates to an injectable material comprising a mixture of synthetic microspheres and platelet-rich blood plasma microspheres for the regeneration of cartilaginous tissue. The presence of plasma microspheres rich in platelets and microspheres of a biodegradable synthetic polymer, facilitates the penetration of stem cells from the microfracture of the subchondral bone, which can make their way between the support microspheres, something that does not occur when the components are not in the form of microspheres, but as a gel, since it cannot be penetrated by the cells until they degrade it.

DESCRIPTION OF THE INVENTION

In this report the expression "platelet-rich plasma or PRP" refers to blood plasma samples with platelet concentrations of double or more than the basal levels that on average supposes 5 x 10 ⁵ or more platelets / pL (platelets per microliter ).

The term "platelet poor plasma or PPP" refers to blood plasma samples with a lower concentration of platelets than platelet rich plasma.

Here, the term "support microspheres" refers to the injectable material, that is, the mixture of platelet rich plasma microspheres and synthetic microspheres.

"Implant", "injectable implant" and "injectable material" may appear in the description of the invention with the same meaning.

A first aspect of the present invention relates to an injectable material comprising a mixture of:

- microspheres of a biodegradable polymer - synthetic microspheres -

- and platelet rich plasma microspheres - PRP microspheres.

In a preferred embodiment, the synthetic microspheres are composed of at least one bio-absorbable polymer: For example, the synthetic microspheres comprise a polymer selected from polylactic acid, polylactic and polyglycolic acid copolymers, polycaprolactone, polyhydroxybutyrate, polyhydroxivalerate, copolymers of the two above, polypeptides, biodegradable polyanhydrides, biodegradable polyurethanes, chitosan, alginate, chondroitin sulfate, hyaluronic acid, starch, dextran and, gelatin, collagen, biodegradable derivatives of cellulose, fibrin, fibrinogen and mixtures of microspheres of several of the above materials.

In addition to the polymers mentioned in the previous paragraph, other synthetic polymers capable of being reabsorbed by the body in periods of less than two years, other bio-absorbable polysaccharides, and other proteins can be used.

In the injectable material of the invention, synthetic microspheres are manufactured by emulsion or microfluidic using methods that have been published in the scientific literature (De la Vega et al. (Uniform polymer microspheres: monodispersity criteria, methods of formation and applications, Nanomedicine 2013 : Vol 8, pp 265-285); DL Elbert, Liquid-liquid two-phase systems for the production of porous hydrogels and hydrogel microspheres for biomedical applications: A tutorial review, Acta Biomaterialia 2011: Vol 7 pp31-56) and M Anindita , P Vilkas, (Drug delivery: Techniques for polymeric microsphere preparation, Research Journal of Biotechnology 2007: Vol 2 pp 58-63)) and are sterilized by the most appropriate method for each material, for example, gamma ray irradiation.

In a particular embodiment, the volume ratio of synthetic microspheres to PRP microspheres is between 0.1 and 80%, more preferably it is in a range between 0.2 and 70%.

Another aspect of the present invention relates to a process for preparing the injectable material defined above, which comprises the following steps:

- obtain PRP microspheres in an oily medium and

- mixing said microspheres with synthetic microspheres.

In a particular embodiment, obtaining the PRP microspheres comprises: a) centrifuging blood taken from a patient,

b) extract a platelet-rich plasma fraction, PRP, and separate and preserve the platelet-poor plasma fraction, PPP,

c) formation of PRP microspheres.

In addition, the process for preparing the injectable material may comprise: d) extracting the PRP microspheres by removing the oily medium by adding a saline medium and centrifuging to separate the aqueous phase containing the PRP microspheres from the oily one,

e) wash the PRP microspheres successively in mixtures of ethanol and sterile saline medium, with increasing concentrations of saline medium until the suspension of the microspheres in the sterile saline medium is finished, or alternatively wash once in acetone before washing in mixtures of ethanol and sterile saline medium.

f) mix the PRP microspheres with the synthetic microspheres before implanting them.

Preferably, the PRP microspheres obtained in step c) have diameters between 30 and 400 microns, more preferably between 50 and 200 microns.

In a preferred embodiment, the saline medium used in step d) is a phosphate buffered saline, PBS (an aqueous saline solution containing sodium chloride, sodium phosphate, potassium chloride, potassium phosphate).

In step c) the PRP microspheres can be formed with platelet activation.

In another embodiment, in step c), the PRP microspheres can be formed without platelet activation.

In a particular embodiment, the formation of PRP microspheres with platelet activation in step c) comprises the following steps:

- add to the PRP of step b) an aqueous solution of a calcium salt, or an aqueous solution of a mixture of the calcium salt and a fibrinogen coagulant enzyme, capable of inducing platelet activation to initiate coagulation of the fibrinogen contained in the plasma and allow the mixture obtained to drip in an oily medium,

- leave the coagular mixture under stirring, obtaining an emulsion at a temperature below or equal to 37 ° C, obtaining the microspheres of PRP.

In a preferred embodiment, the salt used is calcium chloride. In another preferred embodiment, the enzyme used is thrombin.

In a particular embodiment, the oily medium is selected from a mineral oil and a vegetable oil.

The mineral oil can be a paraffinic oil, a naphthenic oil, an aromatic oil or a silicone oil.

Vegetable oil can be olive, sunflower, soy or corn oil.

In a particular embodiment, the formation of PRP microspheres without platelet activation in step c) further comprises obtaining a hydrogel from a precursor of said hydrogel by chemical or enzymatic means.

Through the formation of PRP microspheres, preventing platelet activation, the growth factors they contain are not released before implanting them at the site of regeneration, which could cause them to be lost, in part, during process. For this, the PRP is mixed with a precursor of a hydrogel and the microspheres are formed by a chemical or enzymatic reaction of the hydrogel that does not alter the viability of the PRP platelets. In this way, the PRP is retained in the microspheres containing all its components.

The hydrogel precursor may be hyaluronic acid, gelatin, fibrinogen, chondroitin sulfate, dextran or the PPP extracted in step b).

In a particular embodiment, the formation of the hydrogel by chemical means comprises the following steps:

- modify a hydrogel precursor by reacting with molecules containing acrylate or methacrylate groups, to obtain a modified precursor (PRECmod),

- mixing the PRECmod with the PRP, in PRP / PRECmod dissolution ratios between 5/95 and 50/50 by volume, add a monomer containing acrylate or methacrylate groups such as ethylene glycol methacrylate, ethylene glycol acrylate, hydroxyethyl methacrylate, hydroxyethyl, acrylamides, methacrylamides and with a photoinitiator capable of initiating the reaction by radical route, - forming an emulsion of the product of the previous stage in an oily medium,

- expose the emulsion to ultraviolet light to produce coagulation of the plasma microdroplets that constitute the hydrogel.

Modification of the hydrogel precursor by reaction with molecules containing acrylate or methacrylate groups is performed according to procedures analogous to those described in the references cited in the state of the art (Biomaterials 2005; 26 (15): 2467-77; Macromolecular Bioscience 2009; 9 (12): 1194-201),

The oily medium in which the emulsion is formed can be a mineral oil (paraffinic oil, a naphthenic oil, an aromatic oil or a silicone oil), or a vegetable oil (olive, sunflower, soy or corn oil).

The procedure according to this embodiment continues with steps d), e) and f) above.

In another particular embodiment, the formation of the hydrogel via enzymatic comprises the following steps:

- prepare a solution of a hydrogel precursor with tyramine,

- mix the PRP extracted in step b) with the previous solution in relations

PRP / hydrogel precursor solution between 5/95 and 50/50 by volume, and add horseradish peroxidase,

- emulsion in oily medium with hydrogen peroxide for the time necessary for the formation of the hydrogel.

The preparation of the solution of a hydrogel precursor with tyramine can be carried out following the method described in the document Poveda-Reyes et al. (Reinforcing an Injectable Gelatin Hydrogel with PLLA Microfibers: Two Routes for Short Fiber Production, Macromolecular Materials and Engineering 2015; 300 (10): 977-88);

Preferably, for this embodiment, the hydrogel precursor solution comprises tyramine modified macromolecules that are selected from tyramine conjugates of: gelatin, hyaluronic acid, chondroitin sulfate, dextane and mixture thereof.

The procedure according to this embodiment continues with steps d), e) and f). In the case of the formation of PRP microspheres without platelet activation, both chemically and enzymatically, it is necessary that immediately before implanting the cells in the place where the regeneration is intended, platelets are activated by the addition of calcium chloride and / or thrombin, which causes the fibrinogen of the PRP to coagulate in the form of fibrin and the platelets release their growth factors.

Before mixing the PRP microspheres and synthetic microspheres, the PRP microspheres can be cryopreserved. Prior to mixing both microspheres (synthetic and biodegradable), a process of thawing said cryopreserved PRP microspheres is necessary.

In a particular embodiment, the process further comprises the following steps after obtaining the PRP microspheres in saline medium, after step e):

- remove the saline medium by decantation and aspiration,

- transfer the PRP microspheres to a dimethylsulfoxide solution and the PPP obtained in step b) and freeze the obtained microsphere suspension in liquid nitrogen to cryopreserve until later use,

- defrost the microsphere suspension,

- conditioning by centrifugation and adding PPP to the PRP microspheres.

The PRP microspheres are transferred to a solution of dimethylsulfoxide and the PPP obtained in step b) in which the latter is present in up to 10%, and is frozen in liquid nitrogen, inside cryotubes. In a first stage the suspension of the microspheres is cooled at a speed of up to 2 ° C / min to reach -80 ° C and after 24 hours of stabilization at this temperature, they are introduced into a thermally insulated container, Dewar, containing the liquid nitrogen.

The microsphere suspension is thawed in a 37 ° C water bath for approximately 2 minutes,

Conditioning is carried out by centrifugation at rotation speeds of between 600 and 1000 rpm for 5 minutes to recover the PRP microspheres, adding PPP to the PRP microspheres and centrifuging again between 600 and 1000 rpm for 5 minutes. Finally all the supernatant will be removed. The suspension of defrosted PRP microspheres is mixed with the synthetic microspheres in the saline medium, allowed to decant and the excess liquid is removed.

A final aspect of the present invention relates to the use of the injectable material for the regeneration of articular cartilage.

The injectable material described in this invention has advantages (Figure 7) in comparison with each of the other therapies currently applied in clinical phases or in the market:

It combines the stimulation of the subchondral bone to drive the stem cells to the site of damage and the implantation of a support biomaterial in the form of injectable microspheres that provide growth factors and create the appropriate biomechanical environment for migrant cells to differentiate and originate a cartilage Functional articulation

It does not need the manipulation of cells outside the organism as it happens both in the case of the implantation of autologous chondrocytes and in the case of mesenchymal stem cells that involve very complex processes and facilities, which facilitates the transfer to the clinical application. The techniques of stimulation of the subchondral bone and the extraction and manipulation of PRP are well known in the clinic.

Use as a support for regeneration. The microspheres described herein, once injected, leave migration paths of the stem cells from the subchondral bone. This is an advantage over the implantation of an injectable gel that fills the cartilage defect where regeneration is to occur because in this case the gel structure blocks the passage of new cells from the subchondral bone. The cells in this case must be injected together with the gel precursors, which adds the complexity described in the previous paragraph.

Against the use of macroporous scaffolds, called scaffolds in the scientific literature, as a support for regeneration we have found in the animal model that the microspheres move easily during the growth of the new tissue, which allows it to organize itself in a similar way to the natural one. In the scaffolding pores, growth is restricted by the pores walls. The displacement of the microspheres leaves free space for the newly formed tissue before the synthetic material is bioabsorbed, in this way, materials with long degradation times can be used.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Samples of synthetic and PRP microspheres and their handling or mixing thereof: a) polylactic acid microspheres, PLA, b) mixture of chitosan microspheres with PLA microspheres, c) PRP microspheres and d) mixture of microspheres of PRP with PLA microspheres.

Figure 2. a) FESEM micrograph (field emission scanning electron microscopy) of PLA microspheres with 50 pm scale bar. b) Micrograph performed under an optical microscope of lyophilized and hydrated chitosan microspheres (20x magnification). c) Micrograph performed under an optical microscope of the PRP microspheres after 12 days of cryopreservation (10x magnification). d) SEM micrograph (scanning electron microscopy), with 40 pm scale bar, of the PLA membranes obtained by electro-spinning.

Figure 3. Diagram of the surgery with the implant or (material) of support microspheres injected into a defect of articular cartilage and microfracture of the subchondral bone and a synthetic membrane covers the defect and is attached to the clot covering the defect. Migration of mesenchymal cells (MSC) is shown.

Figure 4. PLA membranes: a) PLA membrane before sterilizing and die-cutting and b) sterilized, die-cut PLA membranes prior to implantation.

Figure 5. Femoral condyles in a rabbit knee model 3 months after surgery a) PLA; b) PLA + PRP; c) surgery control; d) native control.

Figure 6. Rabbit femoral condyles 3 months after fresh surgery and after histological processing and staining with hematoxylin-eosin. Our solution originated a functional tissue with all the characteristics of the articular cartilage that suffered the repaired defect.

Figure 7. Summary table of the advantages offered by the injectable material of the invention and comparison with other regenerative therapies currently in clinical use.

EXAMPLES

Next, the invention will be illustrated by tests carried out by the inventors, in a model of regeneration of articular cartilage in rabbit knee, which demonstrates the effectiveness of the product of the invention.

Example 1

Rabbit blood from the New Zealand breed was centrifuged for 8 minutes at 1800 rpm, separating the PPP and PRP phases. 250 mL of olive oil was introduced into a beaker and a four-blade rod was introduced from a mechanical stirrer. The temperature was controlled at 37 ° C and stirred at 360 rpm. To the PRP was added 5 pL of a 10% aqueous solution of calcium chloride for every 100 pL of PRP and the mixture was dripped onto the oil, maintaining the stirring by controlling the flow of PRP solution by a syringe pump at a rate 0.5 mL / min. Stirring was maintained for 5 hours after finishing adding the PRP solution. After this time the stirring was interrupted, the formed microspheres were allowed to settle and the oil was aspirated. A 5 minute acetone wash was performed with stirring followed by another 5 minute rest and aspirate of the acetone. The obtained microspheres have diameters between 50 and 150 microns (Figure 2c).

Example 2

For the formation of PRP spheres without platelet activation with gelatin hydrogels or hyaluronic acid by enzymatic cross-linking the procedure followed was as follows:

- a solution of the hydrogel precursor was prepared consisting of a 4% solution of gelatin or hyaluronic acid modified with tyramine or mixtures of both containing 50% by mass of each of them in the Krebs Ringer Buffer buffer (formed by 115 mM sodium chloride, 5 mM potassium chloride, 1 mM potassium dihydrogen phosphate and 25 Mm HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid);

- PRP was mixed with this solution in relation to PRP / hydrogel precursor solution 15/85 by volume, adding 10% of the volume of a 12 U / mL solution of horseradish peroxidase and 10% of the volume of a solution. mM hydrogen peroxide;

- the emulsion microspheres were formed in medium consisting of olive oil and the emulsion was maintained 10 minutes (gelation occurs in 5 minutes) for the formation of microspheres,

- subsequently the microspheres were removed by removing the oil with successive steps that included: adding a phosphate buffered saline, PBS (an aqueous saline solution containing sodium chloride, sodium phosphate, potassium chloride, potassium phosphate), centrifuge to separate the aqueous phase containing the PRP microspheres of the oil, aspirating the oil, the operation was repeated once more;

- immediately before mixing the PRP microspheres with the synthetic microspheres, calcium chloride or a mixture of calcium chloride and thrombin was added to the PRP microsphere suspension to activate platelets.

Example 3

In the surgical procedure (Figure 3), the surgeon causes bleeding at the site of the defect by microfracture, nanofracture or drilling of the subchondral bone. The injectable material of the invention is injected and soaked into the blood and is embedded in the blood clot. Next, the defect is covered with a synthetic membrane that is attached to the clot covering the defect (Figures 2d and 4). In a particular application the membrane can be produced by electro-spinning of the same polymers used to produce the microspheres. The entire surgical procedure can be performed by arthroscopy.

The role of PRP microspheres is to release chemotactic and growth factors, which act as a stimulus for the migration and differentiation of mesenchymal cells from the bone to the site of the defect. The role of synthetic, rigid microspheres is to create the appropriate biomechanical environment. In the tests carried out in an animal model of the rabbit knee, it has been shown that, within three months, the PRP microspheres have been reabsorbed while both the membrane and the synthetic microspheres have moved from the implant site to the bone (where they will end up being bio-absorbed), leaving in the place where the cartilage defect was performed a functional tissue with all the characteristics of the articular cartilage (Figures 5 and 6). The regeneration capacity of articular cartilage has been verified with the microsphere implant attached to the microfracture of the subchondral bone in a rabbit knee model in which PLA and PRP microspheres have been injected in a 1/1 volume ratio and He covered the defect with a PLA membrane as shown in Figure 4.

Claims

1. An injectable material comprising a mixture of:

- microspheres of a biodegradable polymer - synthetic microspheres -

- platelet rich plasma microspheres - PRP microspheres

2. An injectable material according to claim 1, wherein the synthetic microspheres comprise a polymer selected from polylactic acid, polylactic and polyglycolic acid copolymers, polycaprolactone, polyhydroxybutyrate, polyhydroxivalerate, copolymers of the two above, polypeptides, biodegradable polyanhydrides, biodegradable polyurethanes , and, chitosan, alginate, chondroitin sulfate, hyaluronic acid, starch, dextran and, gelatin, collagen, biodegradable derivatives of cellulose, fibrin, fibrinogen and mixtures of microspheres of several of the above materials.

3. An injectable material according to claim 1, wherein the volume ratio of synthetic microspheres to PRP microspheres is between 0.1 and 80%, more preferably between 0.2 and 70%.

4. A process for preparing the injectable material defined in one of claims 1 or 2, comprising:

- obtain PRP microspheres in an oily medium and

- mixing said microspheres with synthetic microspheres.

5. A process for preparing the injectable material according to claim 4, wherein the step of obtaining the PRP microspheres comprises:

a) centrifuge blood taken from a patient,

c) formation of PRP microspheres.

6. A method for preparing the injectable material according to claim 5, further comprising: d) extracting the PRP microspheres by removing the oily medium by adding a saline medium and centrifuging to separate the aqueous phase containing the PRP microspheres from the oily one,

7. A process for preparing the injectable material according to claim 5, wherein the PRP microspheres obtained in step c) have diameters between 30 and 400 microns, more preferably between 50 and 200 microns.

8. A process for preparing the injectable material according to claim 6, wherein the saline medium used in step d) is a phosphate buffered saline, PBS.

9. A process for preparing the injectable material according to claim 5, wherein in step c) PRP microspheres with platelet activation are formed.

10. A process for preparing the injectable material according to claim 5, wherein in step c) PRP microspheres are formed without platelet activation.

11. A process for preparing the injectable material according to claim 9, wherein step c) comprises the following steps:

12. A process for preparing the injectable material according to claim 11, wherein the salt used is calcium chloride.

13. A process for preparing the injectable material according to claim 11, wherein the enzyme used is thrombin.

14. A process for preparing the injectable material according to claim 11, wherein the oily medium is selected from a mineral oil or a vegetable oil.

15. A process for preparing the injectable material according to claim 14, wherein the mineral oil is selected from a paraffinic, naphthenic oil, an aromatic oil and silicone oil.

16. A process for preparing the injectable material according to claim 14, wherein the vegetable oil is selected from an olive, sunflower, soybean, corn oil.

17. A process for preparing the injectable material according to claim 10, further comprising obtaining a hydrogel from a precursor of said hydrogel by chemical or enzymatic means.

18. A process for preparing the injectable material according to claim 17, wherein the hydrogel precursor is selected from hyaluronic acid, gelatin, fibrinogen, chondroitin sulfate, dextran and the PPP extracted in step b).

19. A process for preparing the injectable material according to claim 17, wherein the formation of the hydrogel by chemical means comprises the following steps:

- modify a hydrogel precursor by a reaction with molecules containing acrylate or methacrylate groups, to obtain a modified precursor (PRECmod),

- mixing the PRECmod with the PRP, in PRP / PRECmod dissolution ratios between 5/95 and 50/50 by volume, add a monomer containing acrylate or methacrylate groups such as ethylene glycol methacrylate, ethylene glycol acrylate, hydroxyethyl methacrylate, hydroxyethyl, acrylamides, methacrylamides and with a photoinitiator capable of initiating the reaction by radical route,

- forming an emulsion of the product of the previous stage in an oily medium, - expose the emulsion to ultraviolet light to produce coagulation of the plasma microdroplets that constitute the hydrogel.

20. A process for preparing the injectable material according to claim 17, wherein the formation of the hydrogel enzymatically comprises the following steps:

- prepare a solution of a hydrogel precursor with tyramine,

- mix the PRP extracted in step b) with the previous solution in relations

21. A process for preparing the injectable material according to claim 20, wherein the hydrogel precursor solution comprises tyramine modified macromolecules that are selected from tyramine conjugates of: gelatin, hyaluronic acid, chondroitin sulfate, dextane and mix them.

22. A process for preparing the injectable material according to claim 6, further comprising the following steps after obtaining the PRP microspheres in saline medium, after step e):

- remove the saline medium by decantation and aspiration,

- transfer the PRP microspheres to a solution of dimethylsulfoxide and the PPP obtained in step b), and freeze the obtained microsphere suspension in liquid nitrogen to cryopreserve until later use,

- defrost the microsphere suspension,

- conditioning by centrifugation and adding PPP to the PRP microspheres.

23. Use of the injectable material defined in one of claims 1 to 3, for the regeneration of articular cartilage.