WO2015177776A1

WO2015177776A1 - Cell-instructing compositions comprising platelet derivatives

Info

Publication number: WO2015177776A1
Application number: PCT/IB2015/053870
Authority: WO
Inventors: Sara Maria MARQUES DE OLIVEIRA; Rui Luís GONÇALVES REIS; João Filipe COLARDELLE DA LUZ MANO
Original assignee: Association For The Advancement Of Tissue Engineering And Cell Based Technologies And Therapies - A4Tec
Priority date: 2014-05-23
Filing date: 2015-05-25
Publication date: 2015-11-26
Also published as: WO2015177776A4

Abstract

The present application presents cell-instructing compositions rich in platelets derivatives, prepared by layer-by-layer assembling. These cell-instructing compositions may be incorporated in medical devices and be applied in tissue engineering and regenerative medicine.

Description

DESCRIPTION

"CELL-INSTRUCTING COMPOSITIONS COMPRISING PLATELET

DERIVATIVES"

Technical Field

The present application relates to cell-instructing compositions prepared using a layer-by-layer method comprising a platelet derivative, a 2D or 3D substrate and at least one polyelectrolyte for stabilization and/or binding of the platelet derivatives. These cell-instructing compositions may be incorporated in a medical device and be applied in tissue engineering and regenerative medicine.

Background

Platelets are a cost-effective source of multiple bioactive molecules such as adhesive proteins, chemokine, antigen receptors, hemostatic factors and growth factors.

The use of platelet derivatives, i.e., multiple proteins extracted from platelets isolated from blood, has shown huge potential for research and clinical application in tissue engineering and regenerative medicine.

The incorporation of the same on medical devices or/in a controlled manner for the improvement of the bioactive effect is essential.

A successful regeneration induction or cell fate control using platelets derivatives relies on the stabilization, molecular conformation, total content and relative content of its components. As example, the use of high concentration platelet' s derivatives gels inhibits osteoinduction . Osteoinduction has been observed only in certain ranges of concentration. Nowadays there is not a real understand of the bio-molecular mechanism and explanation for this for the best of your knowledge.

There is a high variability associated with the donor platelets regarding the content of specific bioactive molecules or total protein content. The amount of bioactive platelets derivatives for a certain biological function required from each patient may also be a variable to considerer when using this type of elements.

Platelets derivatives have been used as: full gel; foams; gel mixed with other polymers; adsorbed onto polymeric structures (with no specific control over the adsorption) ; antibody-binding for a major attraction of specific components onto a surface or particle. The modification methods already disclosed might limit the amount or the control of platelets derivatives that can be retained on the cell interfaces, the stability and/or release; and might induce conformation changes onto the bioactive molecules. Thus, there is a need to have methodologies that can allow a better control over the adsorption/absorption, incorporation, release profile and presentation of platelets derivatives on medical devices.

Document Leotot J et al . , Platelet lysate coating on scaffolds directly and indirectly enhances cell migration, improving bone and blood vessel formation, Acta Biomaterialia (2013), 9, pages. 6630-6640, discloses that a simple adsorption of platelet lysate onto 3D constructs composed of beta-tricalcium phosphate/hydroxyapatite, can improve bone and vessel formation on those constructs, corroborating the trends observed by other researchers. In this document, the platelet lysate was combined with the constructs by a single adsorption and there is no reference of the execution of such combination recurring to the method of layer-by-layer assembling nor the production of multilayered structures with them. Moreover, this document does not mention the impact that the surface chemistry where platelet lysate where adsorbed could have on the stabilization and control of incorporated proteins.

Document Hua A et al . , Electrostatic layer-by-layer nanoassembly on biological microtemplate : Platelets, Biomacromolecules (2002), 3(3), pages. 560-564 discloses the modification of platelets with silica nanoparticles or bovine immunoglobulin using the layer-by-layer assembling technique. That study focus on the alteration of the surface properties of the platelets with the purpose, e.g., of avoiding platelet aggregation, of blockage of abnormal receptor-agonist binding on their surface, of controlling their content secretion. The document does not relate with the use of platelets or their derivatives as elements of the multilayers prepared by the layer-by-layer assembling technique .

The combination of growth factors with multilayered structures prepared by the layer-by-layer assembling technique has been performed using at least one growth factor as element of one layer in the assembling, or at least one growth factor is loaded in previously prepared multilayered structures, cross-linked or uncross-linked . Document Choi J et al . , Smart controlled preparation of multilayered hydrogel for releasing bioactive molecules, Current Applied Physics (2009), 9, pages. e259-e262, discloses the preparation of hydrogels by the technique of layer-by-layer assembling containing one growth factor but without any reference to the possibility of using a single source of multiple proteins, or platelets.

Document US 8481017 B2 discloses thin films for controlled protein interaction. These thin films for controlled absorption and releasing (controlled by pH and applied potential) are developed by electrostatic interactions. The present invention is based on layer-by-layer assembling not exclusively by electrostatic interactions. For instance, the use of antibody-specific interactions may be beneficial in some embodiments. Our invention is not related to triggered sorbing and/or releasing from or into an aqueous solutions as US 8481017 B2, but instead to create a suitable cell interface/environment by adsorbing the most appropriated content, conformation, and eventually release profile in some embodiments, of platelets derivatives more appropriated for the cell/tissue. In our invention, the stability of the "sorbate" is important since it will affect cell behavior. The "sorbate" will directly interact with cell surface receptors and the sorbate binding stability, the density of sorbate-cell receptor complexes and the interaction time, among others, will define the ability to activate specific signaling pathways and how cells/tissues are affected.

Document WO2010081884 A2 discloses a process for preparing a surface coated by crosslinked polyelectrolyte multilayer films as a biomimetic reservoir for proteins. The object of this disclosure is a process for coating a surface. Is this process the crosslinked polyelectrolyte multilayers film is treated with a protein containing solution, preferably with a growth factor type protein containing solution, as to incorporate said protein on and inside said cross-linked polyelectrolyte multilayers film. This document is based on the modification/treatment of the pre-prepared layer-by- layer films/coatings or coated structures with a growth factor type containing solution.

Document WO2010081884 A2 refers the use of only one growth factor and not mentioning the use of multiple proteins or platelet derivatives as elements for the assembling of the layers, nor as elements to be loaded in previously prepared multilayers .

Document WO 2013/110047 Al discloses multilayer film coating compositions, coated substrates and methods. In some embodiments, a structure includes a first and second layer-by-layer film disposes on a substrate, the structure being characterized in that layer- by-layer removal of at least the second film releases at least one polypeptide, and also may permit release of ions from the ceramic material so that a synergetic effect of the osteoinduction and osteoconductivity of the structure is achieved. The document discloses a growth factor which is selected f rom the group consisting of platelet-derived growth factor (PDGF) , platelet-derived angiogenesis factor (PDAF) , vascular endothelial growth factor (VEGF) , platelet-derived epidermal growth factor (PDEGF) . This document indicates that the growth factor has a specific order in the sequence of the layer-by-layer film (third element) . From WO 2013/110047 Al one understands that one growth factor is chosen from the group mentioned. The film may release at least one polypeptide that was one-by-one selected from the group of growth factors mentioned. The present invention comprises the incorporation of multi bioactive small molecules, polypeptides, among others, from platelets derivatives on a single step in each layer, with no restricted sequence order. In the platelets derivatives, the growth factors mentioned in the WO 2013/110047 Al can be found, among much others. On WO 2013/110047 Al, the individual growth factor (s) is known for some specific function (s) . In the present invention, the final function will depend on whole layer-by-layer structure prepared, even if the same specific growth factor is present on the structure. Depending on the polyelectrolyte used to bind the platelet derivatives, among others setting parameters, for instance, different relative percentages of each individual growth factor will vary and might induce cells to a different fate.

The document WO 2013/110047 Al has never mentioned the advantage of using one unique source containing multiple proteins. Moreover, the growth factor is limited as element in the third layer of the set of tetra-layers .

In summary, none of the documents cited above discloses any strategy capable of incorporating and stabilizing, in a efficiently controlled manner, multiple proteins from platelet derivatives in any 2D/3D device or tissue. The application of the layer-by-layer technique allows a better control of the absorption, incorporation and release profile of the platelet derivatives, simultaneously allowing the presentation/conformation such that the proteins display similar responses as on their natural environment. Additionally, the above cited documents also do not disclose the bio-functionality of the compositions, which is controlled by the properties of the multilayers adjusting the assembling parameters.

Summary

The present application relates to the use of a surface modification technique - layer-by-Layer assembling - to develop cell-instructing compositions based on platelet lysates and derivatives. These compositions may be applied in medical devices for Tissue Engineering and Regenerative Medicine (TERM) . TERM aims to develop materials and strategies to improve, substitute and heal tissues and organs to improving Human health and life.

The present application describes a cell-instructing composition comprising:

- at least one platelet derivative;

- at least one substrate; and

- at least one positive or negative polyelectrolyte for stabilization and/or binding of the platelet derivatives ;

assembled layer-by-layer.

In an embodiment, the platelet derivative is platelet lysate .

In another embodiment, the platelet derivative is a platelet lysate with an initial concentration of 1-2 million platelets/ L, diluted between 1-1500 times, depending on some protein incorporation that may desirable to be improved or reduced; and with a pH between 2 and 11, in order to facilitate incorporation of some protein (s) according to their isoelectric point and subsequent overall charge .

In an embodiment, the polyelectrolyte is alginate, chitosan, carrageenan, and heparin, or combinations thereof .

In another embodiment, the polyelectrolyte is a sulphated polyelectrolyte .

Yet in another embodiment, the previously referred cell- instructing composition comprises a bilayer of platelet lysate and L -carrageenan .

In another embodiment, the previously referred cell- instructing composition comprises 10 tetra-layers of 2x L- carrageenan, chitosan and platelet lysate.

In another embodiment, the previously referred cell- instructing composition is in the form of a film, a coating or a fibrillar structure.

The present invention also describes a layer-by-layer assembling process for obtaining the cell-instructing composition previously described, comprising the following steps :

a) dipping a substrate into a polyelectrolyte solution for at least 30 seconds at 0-60°C;

b) dipping the substrate into a rising solution preferably composed by the same liquid phase of the other solutions for at least 30 seconds between 0-60°C;

c) dipping the substrate into a platelet derivative solution for at least 30 seconds between 0-60°C; d) dipping the substrate into a rising solution preferably composed by the same liquid phase of the other solutions for at least 30 seconds between 0-60°C; or

e) repeating the set of layers between at least 1-6 times .

In another embodiment, the layer-by-layer assembling process comprises steps a) , b) , c) and d) followed by dipping the substrate into a polyelectrolyte solution for at least 30 seconds at 0-60°C, dipping the substrate into a rising solution preferably composed by the same liquid phase of the other solutions for at least 30 seconds between 0-60°C, dipping the substrate into other polyelectrolyte solution for at least 30 seconds at 0-60°C and dipping the substrate into a rising solution preferably composed by the same liquid phase of the other solutions for at least 30 seconds between 0- 60°C, and

repeating the set of layers between at least 1-10 times .

In another embodiment, in the layer-by-layer assembling process, the platelet derivative is platelet lysate.

In another embodiment, in the layer-by-layer assembling process, the polyelectrolyte is alginate, chitosan, carrageenan, and heparin, or combinations thereof. In another embodiment, in the layer-by-layer assembling process, the polyelectrolyte is a sulphated polyelectrolyte .

Yet in another embodiment, the previously described layer- by-layer assembling process comprises at least one additional step selected from: layer-by-layer modification, physical or chemical modification, irradiation, freeze- drying or sterilization.

The present application also describes the use of the previously described cell-instructing composition in regenerative medicine and tissue engineering applications, providing a controlled display of multiple proteins, which are able to synergistically enhance or trigger a specific cell behavior.

The present application also describes a medical device coated with the previously described cell-instructing composition for use in regenerative medicine and tissue engineering applications.

General description

The use of proteins capable of inducing and controlling cell behavior is of greatest interest, e.g. in tissue engineering and regenerative medicine. The combination of two or more proteins, such as growth factors, has shown positive synergistic effects regarding cellular effects, such as, e.g., mitogen, osteogenic differentiation and pro- angiogenic effect. With such purpose, cost-effective sources, autologous, and ready-available, such as human platelets, have been considered quite attractive. The platelet when active release their bioactive content rich in various proteins, such as adhesive proteins, chemokines, antigen receptors, hemostatic factors and growth factors turning them candidates capable of various cellular effects in many different cells.

Successfully inducing regeneration, cell differentiation or achieving cell control using platelet derivatives may be dependent on the stabilization, the conformation, the total protein content, as well as on the content of each of the incorporated protein. For example, high concentration platelet derived gels have been reported to avoid osteoinduction, i.e. the induction of stem cells into bone lineage. On the other hand, osteoinduction could be observed in intermediate ranges of concentrations, which highlights the importance of the controlled incorporation of platelet derivatives in biomaterials .

From the combination of platelet derivatives with layer-by- layer assembled structures, is expected that they stabilize and protect the platelet-derived proteins. The cellular effects of such proteins could be enhanced using reduced quantity to obtain a certain cellular effect.

The reported surface modifications with platelet derivatives have been limiting the content and do not allow their tunable incorporation, nor their stability nor release. Some of those reported approaches may induce conformation changes in the proteins or restrict the essential endocytosis of any protein for the completion of some signaling pathway (e.g., covalently bound proteins) . Therefore, a need exists for methods with better control of the absorption, incorporation and release profile of the platelet derivatives and simultaneously allowing the presentation/conformation such that the proteins display similar responses as on their natural environment.

The present application relates to the use of a surface modification technique - layer-by-Layer assembling - to develop cell-instructing compositions based on platelet lysates and derivatives.

The cell-instructing composition comprises:

a) At least one platelet derivative ;

b) At least one substrate; and

c) At least one positive or negative polyelectrolyte for stabilization and/or binding of the platelet derivatives .

The layer-by-layer assembling technique is commonly known as the alternated deposition of a positive and negative polyelectrolyte, layer by layer, with intermediate washing steps to remove unbound or weakly bound excess polyelectrolyte. There are two main ways to perform layer- by-layer assembling, though not limited to: by immersion or by spraying. By immersion, the structure to be modified is sequentially immersed in polyelectrolyte solution (s), platelet derivative and washing (not with this specific sequence) . Using sprays containing polyelectrolyte ( s ) and platelet derivative, the structure is sequentially sprayed with the solutions. The strategy herein disclosed allows concentrating and/or stabilizing at least one layer of multiple bioactive proteins onto a layer composed by at least one polyelectrolyte/ligand. These layers may be repeated numerous times, with or without other layers rather than the polyelectrolyte and/or the platelet derivative. Thus, the bio-functionality to be incorporated in the device is controlled by the properties of the multilayers adjusting the assembling parameters (e.g., the number of layers and ionic strength of the solutions), the type of platelet derivative and ligand/polyelectrolyte.

The layer-by-layer method process for obtaining the cell- instructing compositions comprises the following steps:

- dipping a substrate into a polyelectrolyte solution of e.g. i-carrageenan for at least 30 seconds at 0-60°C;

- followed by dipping the substrate into a rising solution preferably composed by the same liquid phase of the other solutions for at least 30 seconds between 0-60°C;

followed by dipping the substrate into a platelet derivative solution for at least 30 seconds between 0-60°C;

The refereed set of layers can be repeated at least between 1-6 times or can be followed by dipping the substrate into a polyelectrolyte solution of e.g. i- carrageenan for at least 30 seconds at 0-60°C;

- followed by dipping the substrate into a rising solution preferably composed by the same liquid phase of the other solutions for at least 30 seconds between 0-60°C; - followed by dipping the substrate into a rising solution preferably composed by the same liquid phase of the other solutions for at least 30 seconds between 0-60°C;

followed by dipping a substrate into another polyelectrolyte solution of e.g. chitosan or at least 30 seconds at 0-60°C;

The refereed set of layers can be repeated at least between 1-10 times.

Dipping times and number of cycles must be optimized by testing the effect of the cell-instructing multilayers comprising different layers on cells and/or quantifying the content of proteins incorporated per layer for each application (e.g., differentiation of stem cells into osteoblasts; induction of angiogenesis in endothelial cells; promotion of cell proliferation) .

The type of polyelectrolyte, or other physical-chemical parameters capable of influencing the layer-by-layer assembling may be adjusted (e.g., pH, ionic strength, concentration, polyelectrolyte, the polyelectrolyte degree of sulphation, adsorption time, steps washing, hydrophilic/hydrophobic nature of the layers, etc.) changing the content, conformation, stability and release profile of the bioactive proteins incorporated.

The compositions provided in the present application are capable of incorporating and stabilizing in a controllable manner multiple proteins from platelet derivatives in any 2D/3D device or tissue. The polyelectrolyte term refers to a polymer and/or protein that under certain conditions (e.g., pH) is positively charged (poly-cation) or negatively (polyanion) charged. The polyelectrolyte ( s ) is (are) the ligand of the platelet derivatives, but not exclusively.

According with the polyelectrolyte/ligand used, three different scenarios, or combinations thereof, regarding the adsorption or binding of proteins in the layer (s), can be expected: the type of protein adsorbing may vary; the relative presence of each protein can vary; the conformation of the proteins can vary. Hence the chosen polyelectrolyte may have a significant impact on the mediation of cell behavior by affecting the platelet derivatives incorporated in the multilayers.

The present cell-instructing compositions can be films and and/or coatings and/or fibrillar structures, among others, may be applied in medical devices and have great potential for application in tissue engineering and regenerative medicine of soft and hard tissue. These derivatives are bond and/or stabilized with polymers or other materials during and/or before and/or after the layer-by-layer assembling, and are able to interact with cells.

Brief description of drawings

Without intent to limit the disclosure herein, this application presents attached drawings of illustrated embodiments for an easier understanding. Figure 1 illustrates the variation of thickness of nanocoatings with 6 bilayers of κ-carrageenan/platelet lysate, L -carrageenan/platelet lysate, λ- carrageenan/platelet lysate, heparin/platelet lysate, chitosan/platelet lysate e alginate/platelet lysate respectively, prepared on silicon surfaces and measured by ellipsometry (Specel 2000-vis, Mikropack; n=6; averageisem) .

Figure 2 illustrates the adsorption of proteins from 2 mL of diluted platelet lysate (PL) , onto 6-well plates pre- modified with one layer of different polysaccharides, a) percentage of PL adsorbed and the final surface density, b) percentage of platelet derived growth factor (PDGF) adsorbed and its respective surface density. All pairs of samples were compared using t-test with Welch correction for non-parametric data. Only the ones statistically different (p>0.05) are indicated in the graph with a bar and N.S. (non-significant) . Data represented as average ± sem; n=6.

Figure 3 illustrates the proliferation-fold of hASCs after 4 days in culture in the above-mentioned multilayers rich in platelet lysate (Figure 1, 2, example 1) and respective single layer control and tissue culture polystyrene control (TCPS; proliferation fold considered 1) . Each set of superimposed white bars show the dsDNA content at 20 hours and 4 days (grey) with the n-fold proliferation indicated as bar's inset. ) . Data presented as mean ± SEM

Figure 4 illustrates the staining of extracellular matrix elements of hASCs after 32 days in culture on the mentioned samples, a) Staining with Alizarin Red S (A5533, Sigma- Aldrich) binds to calcium deposits highlighting them with a strong red colour, b) Human osteocalcin stained with green color by immunocytochemistry, nuclei stained with blue (by 4 , 6-DiaKiidino~2-phenyiridole_/ dilactate; D9564, Sigma- Aldrich) and calcium staining with Alizarin Red S. In presence of dexamethasone (Dex) , both calcium and osteocalcin are detected in all the samples. However, in absence of Dex (the osteoinducer ) , a strong presence of calcium and osteocalcin were only detected in the samples with 10 tetra-layers containing platelet lysate (PCL LbL PL) . These observations revealed the importance of a controlled incorporation of platelet derivatives in order to achieve an adequate cell response.

Description of the embodiments

The following optional embodiments are not intended to limit the scope of the present application.

The platelet derivatives are prepared preferably using human platelets or synthetic platelets in the case of human application, otherwise other sources such animal can be used .

Another source of multiple bioactive proteins may be used, such as the ones obtained from decellularized tissue, in vitro cell-produced extracellular matrix, cellular secretomes and other cell lysates.

A platelet derivative is a platelet concentration/dilution or solution of platelet-poor-plasma, platelet-rich-plasma, or Platelet derived Gel, buffer Platelets or platelets treated with solvent, detergent, ions, ultrasounds, thermal cycles, specific antigens or pH' s for the promotion of all, a significant part or specific release of bioactive molecules from the platelets.

Platelet derivatives comprise all compositions resulting from the extraction and/or separation process of at least one, but preferably, multiple bioactive proteins from their cellular compartments. Some of those extraction processes induce their extracellular membrane lysis and the release of the content of the intracellular platelet granules. This has been performed by thermal shocks, mechanical action or platelet activation through the calcium chloride and/or thrombin. In the extraction processes, numerous physical- chemical parameters may be altered adjusting the bioactive composition obtained such as the platelet concentration and the pH. The proteins contained in platelet derivatives have different concentrations; therefore some dilution of the derivative may significantly reduce the amount of one or more proteins reducing their cellular effect while the effect of other (s) may not be altered. Depending on the platelet derivative pH, the global charge or conformation of some proteins may be more efficient to bond the ligant/polyelectrolyte, which may change the cellular effect .

The platelet derivate translate to be is a solution or gel, in which the pH can be varied between 2 to 11 and which is used concentrated or diluted.

The pH of the platelet derivatives can be varied between 2 and 11, depending on the target cells/tissues . Depending on the pH, the different proteins from the derivative will have different net charges and therefore the overall solubility will vary. Varying the pH can also be a method employed to exclude some elements of the platelet' s derivatives. Accordingly to the pH, the most suitable polyelectrolyte to bind the platelets derivatives, more negative or positively charged proteins, shall be selected to the application.

Preferably, the platelet derivative is a platelet lysate with an initial concentration of 1-2 million platelets/ L, diluted between 1-1500 times, depending on some protein incorporation that may desirable to be improved or reduced; and with a pH between 2 and 11, in order to facilitate incorporation of some protein (s) according to their isoelectric point and subsequent overall charge.

The platelet derivatives can be refined for the following ends: the removal of one or more of the elements initial contained; the concentration of one or more of some or specific elements; for the addition of one or more elements that was not initial existent or was in very low concentration; among others.

The platelets derivatives provide several of the following components, among others: Vascular Endothelial Growth Factor (VEGF) ; Fibroblast Growth Factor (FGF) ; Platelet Derived Growth Factor (PDGF) , Insulin Growth Factor (IGF), Transforming Growth Factor (TGF) , Epidermal Growth Factor

(EGF) , Bone Morphogenic Protein (BMP), Interleukin (IL: Ira, 2, 4, 6, 7, 8, 9, 10, 13), Eotaxin, Osteocalcin, Osteonectin, Vitronectin, Fibronectin, P-selectin, Trombospondin, Platelet factor-4 (PF4), Nerve Growth Factor

(NGF) , Hepatocyte Growth Factor (HGF) , Tumor Necrosis Factor (TNF) , Interferon, Granulocyte colony-stimulating factor (GSF) , Stem Cell Growth Factor (SCGF) , Leukemia inhibitory factor (LIF) , Macrophage Inflammatory Protein (MIP) , Monocyte Chemotactic Protein (MCP) , Growth Regulated Oncogene (GRO) , Platelet endothelial cell adhesion molecule-1 (PECAM-1) .

More specifically, the platelet lysate is a complex cocktail of growth factors (e.g., VEGF, FGF, PDGF) , hemostatic factors (e.g., fibrinogen, Protein S), adhesive proteins (e.g., fibronectin, vitronectin, thrombospondin, vitamin D-binding protein), antigen receptors (e.g., P- selectin, PECAM) , chemokine (e.g., IL-8, IL-lbeta, PF-4, CCL2 SDF-lalfa) and small molecules (e.g., ions Ca2+, serotonin, histamine, epinephrine)

The platelet derivative is absorbed or adsorbed before and/or during the assembly process and/or after as finishing step.

The substrate may be a 2D or 3D substrate with any geometry, a living tissue, cells or single cell and is the structure where the layer-by-layer with platelet derivatives, among others, will be applied.

The material-based substrate may be flat, topographically or geometrically controlled or even randomly prepared.

The substrate may be a biological substrate or (e.g., tissues; cells) or a non-biological one (e.g, polycaprolactone ; bone cements) .

The substrate may also be composed of one or more materials, or mixtures from the categories: natural or synthetic proteins (e.g. silk fibroin, synthetic peptides, recombinant proteins), natural polymers (e.g., chitosan, alginate, carrageenan) , polymers derived from natural compounds (e.g., Polylactic acid), synthetic polymers (e.g., polycaprolactone, polyethylene), metals (e.g., titanium, aluminium and vanadium, metallic blends such as titanium-aluminum-vanadium) , ceramics from natural or synthetic origin (e.g. calcium phosphates, biological derived (e.g, decellularized tissue matrices rich in ceramics of autologous, allogenic, xenogenic or in vitro source, among others.

Polyelectrolytes are those charged polymers used to attract proteins from the platelets derivative. The sequence of the multi-layered structure can be any. The polyelectrolytes used for the binding of platelet' s derivative will affect the parts adsorbed from it, the content and stability. Therefore, it should be selected according with the targeted cell behavior.

Polyelectrolytes or platelets derivatives binding agents can be used from the following categories: natural derived (e.g., alginate, chitosan, carrageenan, ulvan, fucoidan, gellan gum, cellulose), animal origin (e.g., chondroitin sulphate, heparin), synthetic or modified ones (e.g., sulfonated or sulphate natural or synthetic materials; polymers modified with heparin-like affinity groups); antigen immobilization for an increased binding of a specific protein; non or sulphated/sulfonated micro/nanoparticles ; nano/micro particles with antigen immobilized for an increased binding of a specific protein from platelets derivatives, avidin, avidin-biotin, antibodies, biotinated antibodies, combinations of the refereed categories in at least one layer, among others.

Depending on the polyelectrolyte used three different scenarios are expected for the absorption: different growth factors or other bioactive proteins might be adsorbed; different ratios between the different growth factors or other bioactive proteins; different conformations of the bioactive factors.

Preferably the polyelectrolyte is a sulphated polyelectrolyte. More preferably, the polyelectrolyte is a sulphated polyelectrolyte of marine origin, such as alginate, chitosan, carrageenan, and heparin-like compounds .

The carrageenan may be selected from κ-, ι- λ-, β-, γ-, δ-, -, μ-, ν-, θ-carrageenan or other chemically modified molecular structures.

The cell-instructing compositions of the present application can be films and and/or (nano ) coatings and/or fibrillar structures, among others.

A 2D application of the cell-instructing compositions is nanocoating which may comprise a bilayer of polyelectrolyte and platelet lysate, wherein the polyelectrolyte is i- carrageenan .

A 3D application of the cell-instructing compositions may comprise around 10 tetra-layers of polyelectrolytes and platelet lysate, wherein the polyelectrolytes are i- carrageenan and chitosan, comprising 10 tetra-layers of 2x L -carrageenan, chitosan and platelet lysate.

The fibrillar structures are often created in 3D structures to increase cell adhesion points and simultaneously improve and control the biochemical 3D environment. Depending on the stage where the layer (s) containing the platelet derivatives is (are) carried out: before, simultaneously or after the layers that introduce small polyelectrolyte complexes in the modified 3D structure, the release profile and the type of proteins incorporated into the multilayers may vary. Alternatively, platelet derivatives can be embedded and adsorbed/absorbed on multilayers a priori containing or in absence of additional platelet derivative.

The fibrillar structures containing the platelet derivatives can also be prepared. To create the fibrillar structures it is used higher concentration and lower rising times than usual Layer-by-Layer assembling, permitting a higher mass deposition and the formation of small complexes that when frozen and freeze-dried are shaped to fibrillar structures. The platelet derivative can be absorbed/absorbed onto the coatings and small complexes; low concentration layer-by-layer with platelet derivative can be performed after creating the fibrillar structures; the platelet derivative can be included during the layer- by-layer for the small complexes formation (high concentration of polyelectrolytes ) ; among other sequences.

Layer-by-Layer can be performed by any of the sub^¬ categories available, among others: dipping layer-by-layer; spraying layer-by-layer; Solid free form fabrication machine assisted dipping or spraying layer-by-layer. The layer-by-layer assembling process may comprise alternated or simultaneous immersion and/or spraying of the polyelectrolyte and platelet derivative preferably alternated with at least one step of dipping or spraying with rinsing solution.

After Layer-by-layer modification, the 2D/3D structure or living tissue may be subjected to finishing steps e.g.: one or more layer-by-layer modification; physic-chemical modification; irradiation; freeze-drying; sterilization, among others .

In some embodiments, an acellular or cellular medical device is coated or structure with the platelet derivatives, or other similar cellular derivatives.

Those are bound and/or stabilized with other polymers or materials, and are able to interact and bind to cells, and/or releases the content of the platelets derivatives incorporated in the multilayered structure.

These derivatives are bond and/or stabilized with polymers or other materials during and/or before and/or after the layer-by-layer assembling.

Examples

Example 1 - 2D Coatings rich in platelet lysate

24-well low adhesion culture plates (CLS3473, Sigma- Aldrich) , previously aminated with a solution of 0.5% (v/v) polyethyleneimine ( Sigma-Aldrich, P3143) for 8 to 12 hours and extensively washed with distilled water, were modified with various multilayer combinations of polysaccharides with platelet lysate. The polysaccharides used were chitosan (average molecular weight and deacetylation 80%, MKBB0566; Sigma-Aldrich) , κ-carrageenan ( Sigma-Aldrich, 22048), L -carrageenan (Fluka, 22045), λ-carrageenan (Sigma- Aldrich, 22049) , sodium heparin (Sigma-Aldrich, H3149) and sodium alginate (Sigma Aldrich, 250 cP) .

Platelet lysates were obtained by thermal lysis (3 cycles of temperature: -196°C and +37°C), and then centrifuging at 1400g for 10 min the platelet-rich plasma was obtained from different donors with platelet count (COULTER® LH 750 Hematology Analyzer) of 1 million/ L. Platelet lysates were diluted 100 times in the same buffer of the respective polysaccharide-pair : 1 M sodium acetate pH 6 with 40 mM sodium chloride (in case of chitosan) or 1M trisaminomethane-HCL pH 7.4 with 40 mM sodium chloride (in other cases) . The polysaccharide solutions were prepared at a concentration of 0.5 mg/mL.

The layer-by-layer assembling was initiated by adsorbing the negative polysaccharide. In the case of chitosan, the first layer was created using 0.5 mL of alginate for 4 minutes. The solutions were removed from the wells and 0.5 mL of wash solution was placed during 30 seconds to remove unbound or weakly bound polyelectrolyte . The washing step was repeated. In the case of the multilayer containing chitosan, 0.5 mL thereof was placed in the wells for 4 minutes followed by the same washing step. Then, 0.5 mL of platelet lysate diluted in the same buffer of the polysaccharide-pair, were adsorbed into each well for 10 minutes. After, the wells were washed according to the washing step described above. Hence, with a bilayer (polysaccharide/platelet lysates xl) completed, this set was repeated five more times, corresponding to nanostructured and multilayered coatings rich in platelet derivatives containing 6 bilayers, prepared by dipping layer-by-layer assembling.

The ability of each polysaccharide to adsorb proteins from platelet lysate and the platelet-derived growth factor (PDGF) were measured. To perform those measurements, 6-well plates were coated with the polysaccharides as above- mentioned using volumes of 2 mL . Thereafter, platelet lysates were adsorbed for 30 minutes, after which the solution was reserved for the measurements. Each well was washed with the same buffer of the polysaccharide solution and its volume was also reserved to measure the unbound proteins. Total protein was quantitated using a spectrophotometer (NanoDrop 1000 Spectrophotometer, Thermo Scientific) . The absorbance of the lysed platelets before and after adsorption, and of the washing solution of each well was measured on a 2 \i droplet at a wavelength of 280 nm. PDGF was quantified using an ELISA kit (900-M04, Prepotech) following the supplier's instructions. Absorbance was measured using a microplate reader (Synergy HT, Bio-Tek Instruments) at normalized wavelength of 450 nm. The results indicate that there is a large change in the adsorption of total protein and PDGF depending on the polysaccharide. Thus, according to the proteins of interests, some polyelectrolytes may be more suitable than others . 5,000 human adipose derived stem cells (hASCs) suspended in basal medium (alpha MEM medium, 12000-063, Alfagene) , were dripped into each well. The wells were modified with the PL rich multilayers or with a single layer of polyelectrolyte . The well-plates were incubated for 20 hours or 4 days after which the total double strand DNA (dsDNA) was quantified in order to assess cell adhesion and proliferation. dsDNA was quantified using the Quant-iT™ PicoGreen® dsDNA assay kit (Molecular Probes/ Invitrogen) . Cell proliferation fold variation was calculated by assuming 1-fold the difference of dsDNA amount from 4 days and 20 hours in the tissue culture polystyrene (TCPS) . The differences were calculated for all the samples and assuming the difference in TCPS is 1-fold, all the others were calculated applying the Rule of Three .

It was observed that some polyelectrolytes are more efficient for the incorporation of proteins from platelet lysate that are capable of promoting cell proliferation (e.g., L -carrageenan more than alginate, chitosan or λ- carrageenan) .

Example 2 - 3D scaffolds with coatings and fibrillar structures rich in platelet lysates

3D structures of polycaprolactone (PCL; Mw 70000-90000, 440 744, Sigma-Aldrich) , pre-aminated with 10% (v /v) ethylenediamine (E1521, Sigma-Aldrich) in 2-propanol (20842.330 VWR) for one hour at 37°C and extensively washed with distilled water, were modified with coatings and fibrillar structures by layer-by-layer assembling with platelet lysates pursued by freeze-drying . These PCL 3D structures (-0.5x0.5x0.4 cm) with 0.5 mm filament distance, alignment of 90° and 0.3-0.4 mm layer thickness (total 10 layers) were prepared with a Bioplotter™ (Envisiontech, Germany), using a 22G hypodermic needle (Z103837, Sigma- Aldrich) .

The polysaccharides selected were: chitosan (degree of deacetylation 80%, MKBB0566; Sigma-Aldrich) , and L- carrageenan (Fluka, 22045) . Both were dissolved in 1M sodium acetate 40 mM pH 5.5 sodium chloride and prepared at a concentration of 4 mg/mL. Initially, two bi-layers of carrageenan and chitosan were assembled onto the aminated 3D PCL, interspersed with several extensive rinsing with the same buffer.

Platelet lysates were prepared as mentioned in Example 1, and diluted with the same buffer solution of the polysaccharides. Platelet lysates were adsorbed between layers of carrageenan, whose layer set was repeated n times: ( carrageenan-platelet lysates (or chitosan) - carrageenan-chitosan) _n . The steps of adsorption of polysaccharides, as well as the wash steps had duration of 4-5 minutes each, while the steps for the incorporation of platelet lysates had duration of 10 minutes. The prepared samples were: PCL (unmodified); PCL LbL (PCL modified with (carrageenan-chitosan-carrageenan-chitosan) io ) ; PCL LbL PL (PCL modified with (carrageen-platelet lysate-carrageenan- chitosan) io ) ; and PCL LbL PLx3 (PCL modified with (carrageen-platelet lysate-carrageenan-chitosan) ₃₀) . To complete the formation of fibrillar structures, the modified 3D structures were washed with distilled water, frozen at -80° and freeze-dried during 1 to 2 days. hASCs were cultured on those samples during 4 days in basal medium (alpha MEM medium, 12000-063, Alfagene) , followed by 28 days in culture with osteogenic supplements (with 10^~8 M dexamethasone, 10 mM beta-glycerophosphate and 50 mg/mL L- ascorbic acid) or osteocondutive supplements (no dexamethasone, 10 mM beta-glycerophosphate and 50 mg/mL L- ascorbic acid) .

After the culture period, it was found that the presence of platelet lysate-rich multilayers has not interfered with the osteogenic differentiation upon culture with standard osteogenic differentiation media. With the culture in absence of dexamethasone, it has observed that the multilayers rich in platelet lysate with 10 tetra-layers (PCL LbL PL) were capable of inducing the differentiation of hASCs into osteoblasts depositing osteocalcin accompanied by a mineralized matrix. On the other hand, with 30 tetra-layers (PCL LbL PLx3) the differentiation was inhibited: no calcium nor osteocalcin were detected in the extracellular matrix. Therefore, the controlled incorporation of platelet lysate into 3D structures is essential to achieve an osteo-inductive effect onto hASCs.

Naturally, the present embodiments and examples are not in any way limited to the embodiments and examples described in this document and a person with average knowledge in the field will be able to predict many possible changes to it without deviating from the main idea, as described in the claims .

Claims

Cell-instructing composition comprising

at least one platelet derivative;

at least one substrate; and

at least one positive or negative polyelectrolyte for stabilization and/or binding of the platelet derivatives ;

assembled layer-by-layer.

Cell-instructing composition according to claim 1, wherein the platelet derivative is platelet lysate.

Cell-instructing composition according to claim 2, wherein the platelet derivative is a platelet lysate with an initial concentration of 1-2 million platelets/ L, diluted between 1-1500 times, depending on some protein incorporation that may desirable to be improved or reduced; and with a pH between 2 and 11, in order to facilitate incorporation of some protein (s) according to their isoelectric point and subsequent overall charge.

Cell-instructing composition according to any one of the previous claims, wherein the polyelectrolyte is alginate, chitosan, carrageenan, and heparin, or combinations thereof.

Cell-instructing composition according to claim 4, wherein the polyelectrolyte is a sulphated polyelectrolyte .

6. Cell-instructing composition according to any one of the previous claims, comprising a bilayer of platelet lysate and L -carrageenan .

7. Cell-instructing composition according to any one of the previous claims, comprising 10 tetra-layers of 2x L -carrageenan, chitosan and platelet lysate.

8. Cell-instructing composition according to any one of the previous claims, wherein said composition is in the form of a film, a coating or a fibrillar structure.

9 . Layer-by-layer assembling process for obtaining the cell-instructing composition described in claims 1-8, comprising the following steps:

c) dipping the substrate into a platelet derivative solution for at least 30 seconds between 0-60°C;

d) dipping the substrate into a rising solution preferably composed by the same liquid phase of the other solutions for at least 30 seconds between 0-60°C; or

e) repeating the set of layers between at least 1- 6 times.

10. Layer-by-layer assembling process according to claim 9, comprising steps a) , b) , c) and d) followed by

dipping the substrate into a polyelectrolyte solution for at least 30 seconds at 0-60°C, dipping the substrate into a rising solution preferably composed by the same liquid phase of the other solutions for at least 30 seconds between 0-60°C, dipping the substrate into other polyelectrolyte solution for at least 30 seconds at 0-60°C and dipping the substrate into a rising solution preferably composed by the same liquid phase of the other solutions for at least 30 seconds between 0-60°C, and repeating the set of layers between at least 1- 10 times.

11. Layer-by-layer assembling process according to claim 9, wherein the platelet derivative is platelet lysate.

12. Layer-by-layer assembling process according to claim 9, wherein the polyelectrolyte is alginate, chitosan, carrageenan, and heparin, or combinations thereof.

13. Layer-by-layer assembling process according to claim 12, wherein the polyelectrolyte is a sulphated polyelectrolyte .

14. Layer-by-layer assembling process according to any one of claims 9 to 13, comprising at least one additional step selected from: layer-by-layer modification, physical or chemical modification, irradiation, freeze- drying or sterilization.

15. Cell-instructing composition according to any one of claims 1-8 for use in regenerative medicine and tissue engineering applications, providing a controlled display of multiple proteins, which are able to synergistically enhance or trigger a specific cell behavior .

16. Medical device coated with the cell-instructing composition described in any one of claims 1-8 for use in regenerative medicine and tissue engineering applications .