PLATELET LYSATE FOAM FOR CELL CULTURE, CELL THERAPY AND TISSULAR REGENERATION AND METHOD FOR OBATINING SAME

Abstract

The present invention relates to a platelet lysate foam obtained from blood derivative (allogenic or autologous) which retains the biological properties of the platelet lysate and has optimal properties, in particular mechanical but also storage, which allow sale thereof and make handling thereof easier.

The present invention also relates to the use of a platelet lysate foam for therapeutic purposes, cell culture and cell therapy.

The present invention also relates to a process for getting a platelet lysate foam by a process of drying in a supercritical CO.sub.2 atmosphere.

Claims

1. A platelet lysate foam characterized in that it comprises TGF-β, EGF, PDGF-AB, IGF-1, VEGF and bFGF within a polymerized fibrin matrix.

2. The platelet lysate foam according to claim 1, further comprising calcium and/or tranexamic acid.

3. The platelet lysate foam according to claim 1, characterized in that said foam has a porosity of between 70% and 95%.

4. A process for making a platelet lysate foam comprising the steps of: providing a hydrogel by polymerization of a platelet lysate; substituting aqueous solvent with a polar solvent by washing; and then drying by a drying process in a supercritical CO.sub.2 atmosphere.

5. The process according to claim 4 characterized in that the hydrogel is obtained by polymerization of a platelet lysate, where said platelet lysate is combined with a polymerization initiator, with an agent with which to maintain the isotonicity and the swelling of the gel, and with a coagulation stabilizer.

6. The platelet lysate foam obtained by the process of claim 4.

7. A method of cell culture, comprising p1 culturing cells in the platelet lysate foam according to claim 1.

8. A method of supporting skin healing and regeneration of the dermis, for supporting osteogenesis and bone regeneration, and/or for tissue regeneration and/or cell therapy in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the platelet lysate foam of claim 1.

9. A method of treating corneal disorders in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the platelet lysate foam of claim 1.

10. A method of supporting osteogenesis and bone regeneration, or for supporting periodontal tissue regeneration in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the platelet lysate foam of claim 1.

11. The platelet lysate foam according to claim 3, wherein the foam has a porosity of about 80%.

12. The platelet lysate foam according to claim 5, wherein the polymerization initiator is calcium chloride (CaCl.sub.2), thrombin, and/or genipin, the agent to maintain isotonicity and swelling is sodium chloride (NaCl), and the coagulation stabilizer is tranexamic acid, amino-caproic acid and/or fibronectin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0225] Other characteristics, details and advantages the invention will appear upon reading the following detailed description, and analyzing the attached drawings, on which:

[0226] FIG. 1 shows the compression strength of the platelet lysate foams according to the invention (“Foams”), in comparison with the initial hydrogels (“Hydrogels”) (n=12);

[0227] FIG. 2 shows the breakdown kinetics in aqueous medium of the platelet lysate foam according to the invention;

[0228] FIG. 3 shows the release of VEGF growth factor (in pg/mL) over time (in days) as a function of the various forms (platelet lysate foam according to the invention, platelet lysate hydrogel and control liquid).

EXAMPLES

Example 1

Getting Hydrogel from Platelet Lysate

[0229] Platelet lysate hydrogels were obtained from platelet lysate combined with various elements in liquid form according to the proportions as summarized in Table 1 below:

TABLE-US-00001 TABLE 1 Constituents Proportion (%) Platelet Lysate between 60 and 80% CaCl.sub.2 between 2 and 3% NaCl between 20 and 30% Tranexamic acid between 0.1 and 0.5%

[0230] The resulting hydrogels have optimal fibrous and porous structures, in particular for promoting cell proliferation, migration and differentiation.

[0231] Advantageously these platelet lysate hydrogels are used for obtaining platelet lysate foams capable of providing the same properties as the platelet lysate while also demonstrating superior qualities for sale. The use of platelet lysate foams is easier and may be suitable to all pathologies treated by tissue or cell engineering.

Example 2

Process for Getting the Platelet Lysate Foam

[0232] The platelet lysate hydrogel is then dried in the reaction vessel of a supercritical CO.sub.2 dryer. This type of reaction vessel advantageously allows maintaining the three-dimensional structure of a hydrogel during the drying operation.

[0233] In order to extract the water contained in the platelet lysate hydrogel, it is soaked for 48 hours in an acetone bath and then separated from the support thereof before being placed in the closed reaction vessel of the dryer. Preferably, the hydrogel is soaked in a glass or metal container.

[0234] The temperature of the reaction vessel chamber is lowered to a temperature below 10° C. in order to allow liquid CO.sub.2 to enter. The reaction vessel is filled with the liquid CO.sub.2 until immersing the samples and then the assembly is left to soak for 45 minutes in order to allow the liquid CO.sub.2 to penetrate the porous network of the gel. Rinsing is then done by emptying the CO.sub.2 present in the chamber and then letting in the same new quantity of liquid. There soaking/rinsing operation is repeated three times. After the cycles, the reservoir is again filled halfway, the reaction vessel closed and then the temperature is progressively raised to 40° C. and the pressure up to 90 bar. Since the reaction vessel was closed, when the temperature increases, the pressure inside the reaction vessel increases. The supercritical state, which corresponds to the fourth state of matter, is reached when the temperature is over 31° C. and the pressure over 74 bars. The reaction vessel was held at that temperature and that pressure for four hours and then rapidly degassed and depressurized over 90 seconds.

[0235] All of the acetone present in the hydrogel was replaced by liquid CO.sub.2 during the soaking/rinsing phases, and then when increasing the temperature and pressure, any trace of solvent is eliminated, the fiber network is then dry and the dry gel has a porous, fibrous foam form.

[0236] As shown in the following examples, the resulting platelet lysate foam advantageously keeps the three-dimensional fibrous arrangement thereof (example 3) and the major elements such as sodium, chlorine, phosphorus, sulfur and calcium (example 4).

[0237] The platelet lysate foam additionally has better mechanical properties than those of the initial hydrogel (example 5). The resulting foam is a dry material capable of being easily stored and rehydrated (example 6), which supports the rapid penetration of biological fluids and cells but also the cell activity by releasing growth factors and other proteins (example 7).

Example 3

Characterization of the Microstructure

[0238] The fiber network of the platelet lysate foam was observed under environmental scanning electron microscope with metallization before and after drying with supercritical CO.sub.2.

[0239] The process serves to get a fibrous network such as a three-dimensional matrix. Advantageously, the fibrin network retains the three-dimensional fibrous arrangement thereof.

[0240] The mesh of the fibrous network is larger after drying which allows checking the porosity. It is thus possible to change the diffusion phenomena inside the porous material by modifying the average porosity and average diameter of the pores predominantly present in the three-dimensional network.

[0241] Thus, in that way it is possible to change the penetration of fluids and cells (and also the growth factor release kinetics).

[0242] These two parameters change the growth factor release kinetics and of anything which may have been incorporated in the foam. This does not change the quantity released but the speed at which the growth factors are going to be released and likewise, the duration of action of the foam.

[0243] In general, the speed of release increases when the average porosity increases and when the average size of the pores increases.

[0244] The porosity of the platelet lysate foam was quantified and the porous network was characterized.

[0245] The method used is mercury porosimetry (device: Autopore III, Micromeritics). The method consists of having the mercury enter into the pores of the platelet lysate foam under increasing pressure. The platelet lysate foam sample is going to be weighed in a conductance cell before and after filling with mercury. An analysis of the mercury pressure differential is going to be done in order to quantify the porosity and characterize the porous network.

[0246] Advantageously, the platelet lysate foams according to the invention have an average porosity of around 80%. Advantageously, an average porosity of around 80% allows fluids, molecules, ions and cells to get in between the fibers of the network and thus support penetration thereof.

[0247] The diameter of the pores predominantly present in the platelet lysate foam is 3.5 μm. Advantageously, this diameter of pores predominantly present allows fluids, ions, molecules and cells in the environment to enter all the way into the core of the network.

[0248] It is observed that a minority of the pores have an average diameter included between 10 and 11 μm, some pores have a diameter included between 6.5 and 8 μm, and pores have an average diameter included between 0.4 and 2 μm.

Example 4

Characterization of the Mechanical Properties

[0249] TAX T2 compression tests were done in order to characterize the mechanical properties of the platelet lysate foams dried with supercritical CO.sub.2. These mechanical properties were compared to those of the initial hydrogels (“hydrogels” in FIG. 1).

[0250] The following were the conditions: [0251] Loading speed: 2 mm/min; [0252] Analysis of the behavior up to 60% compression; [0253] Device: TA.XT Plus Texture Analyzer.

[0254] The dry networks have a distinct increase of their compression strength compared to the initial hydrogels (n=4; p<0.01).

[0255] The platelet lysate foams according to the invention therefore have better mechanical properties than those of the initial hydrogel. These foams can thus be easily handled with forceps or by hand without disintegrating as the hydrogel does.

Example 5

Determination of the Rehydration Rate after Drying

[0256] The rehydration rate after drying the platelet lysate foams according to the invention was determined. The method used is the weighing method.

[0257] The following are the conditions: [0258] The samples were soaked in 1200 μL of water at 25° C. for 48 hours; [0259] The rehydration rate is calculated using the formula:

[00001]$\begin{array}{cc}\mathrm{rehydraton}\mathrm{rate}=\frac{\left(\mathrm{wet}\mathrm{weight}-\mathrm{dry}\mathrm{weight}\right)}{\mathrm{dry}\mathrm{weight}}\times 100& \left[\mathrm{Math}.1\right]\end{array}$

[0260] The average rehydration rate calculated is 804.9%.

[0261] The platelet lysate foam therefore has a significant rehydration rate. Also advantageously, and in the absence of water, the platelet lysate foam has a favorable storage for sale thereof. In fact, and in the absence of water, the dry material does not break down over time.

Example 6

Breakdown and Extended Release Kinetics

[0262] The breakdown kinetics in aqueous medium and release of a growth factor included in the platelet lysate foam were evaluated.

[0263] Breakdown kinetics in aqueous medium

[0264] The method used is the weighing method.

[0265] The following are the conditions: [0266] Samples soaked in 20 mL of water at 25° C.; [0267] Tracking of the breakdown by weighing of the material.

[0268] As shown in FIG. 2, the platelet lysate foam according to the invention disintegrates at the end of five days. The growth factors therefore have an extended release and are not released immediately as is the case with the platelet lysate liquid.

[0269] Release of a Growth Factor, VEGF

[0270] The VEGF was assayed in order to evaluate the release thereof. The method used is that of the Human VEGF Pre-Coated ELISA Kit test from Biogems. The release of the VEGF by the platelet lysate foam according to the invention was compared with the release kinetics of the platelet lysate hydrogel. A liquid was used as control, as is shown in FIG. 3.

[0271] The platelet lysate hydrogel was prepared by the process described in example 1 and the platelet lysate foam was prepared by the process described in example 2.

[0272] The measurement of the absorbance was done at 450 nm.

[0273] As shown in FIG. 3, the VEGF is released progressively over five days until reaching the maximum concentration thereof. The extended release of VEGF continued for 25 days.

[0274] In that way, and advantageously, the platelet lysate foam according to the present invention initially composed based on platelet lysate rich in growth factors, released VEGF over time. That shows that the growth factors are encased in the fibrin network and are accessible to the cells.

[0275] This release is extended and in similar quantity to that of the platelet lysate hydrogel, confirming there is no loss of protein material during the drying process.

[0276] Therefore advantageously, the platelet lysate foams according to the invention may be used in many biological applications such as regeneration and repair of damaged tissues.

[0277] In fact, the natural presence of growth factors and cytokines such as VEGF, PDGF, EGF and TGFβ which are released during implantation in the medium of the platelet lysate foam according to the invention, thus contributing to the growth of tissues and the development of organs, constitutes an important argument for the biomedical use of platelet lysate foams according to the invention.

[0278] Beyond the advantage thereof from extended release compared to the platelet lysate hydrogels, the platelet lysate foams according to the invention are further easier to handle and have improved storage and mechanical properties.