• 专利附录
  • 专利说明
  • 权利要求
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    • 专利摘要:
    Procedure for obtaining a prosthesis coated with a functional coating and cells adhered to said coating, as well as the coated prostheses obtained by this procedure. A method for obtaining a coated prosthesis comprising the following steps: depositing a functionalized sheet with reactive groups on at least part of the surface of a prosthesis, where deposition takes place by means of the activated steam silanization technique; immobilize at least one organic molecule in the previously deposited sheet to obtain a first (functional) coating; and culturing at least one type of cells on the first coating in vitro, under conditions that allow survival, cell adhesion and, preferably, differentiation to lineages compatible with the host tissue, resulting in the prosthesis with a second (biological) coating comprising cells adhered to the first (functional) coating. As well as the coated prosthesis obtained by this procedure. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2732949A1
    申请号:ES201930949
    申请日:2019-10-29
    公开日:2019-11-26
    发明作者:Rigueiro Jose Perez；Tortuero Gustavo Víctor Guinea；Nieto Daniel Gonzalez；Gomez Milagros Ramos
    申请人:Universidad Politecnica de Madrid；
    IPC主号:
    专利说明:

    [0001]
    [0002]
    [0003] FUNCTIONAL COATING AND CELLS ADHERED TO SUCH COATING, AS WELL AS THE COVERED PROSTHESES OBTAINED BY THIS PROCEDURE
    [0004]
    [0005] TECHNICAL SECTOR
    [0006]
    [0007] The invention is part of the field of biomaterials and tissue engineering and, in particular, in the procedures developed to improve the biocompatibility of these materials, by obtaining a prosthesis or implant that has on its surface a coating formed by molecules organic, in particular pharmaceutical active compounds or biomolecules, and cells.
    [0008]
    [0009] More specifically, the process described herein allows obtaining a prosthesis or implant having a functional coating comprising a functionalized sheet with reactive groups and organic molecules capable of interacting with said sheet, in particular pharmaceutical active compounds or biomolecules; and cells adhered to this functional coating. Thus, the prosthesis or implant obtained by this procedure comprises cells of a predetermined cell lineage before being implanted in the recipient organism.
    [0010]
    [0011] These prostheses or implants can be used in the medical and veterinary field.
    [0012]
    [0013] STATE OF THE TECHNIQUE
    [0014]
    [0015] The objective of increasing the life expectancy of the population and achieving more and more advanced ages in the best conditions implies the maintenance of a sustained effort in the development of new therapies that allow to solve pathologies until now without treatment, or that improve the treatments available today. As a consequence of this effort, the study of biomaterials, in particular, those that can replace damaged organic tissues by wear or trauma, has acquired a notable role. This prominence is related to the possibility offered by the use of new biomaterials to design therapies beyond the traditional approach based on the exclusive use of drugs or, even, the most novel in that drugs are combined with cells. In this context, improving the biocompatibility of the metal, ceramic and polymeric biomaterials currently available represents one of the main challenges.
    [0016]
    [0017] The paradigm currently accepted to explain the biocompatible behavior of a material or not assumes a process of interaction between the organism and the implant that develops in two stages (Kao WJ, Evaluation of protein-modulated macrophage behavior on biomaterials: designing biomimetic materials for cellular engineering, Biomaterials, 20 (1999) 2213-2221). In the first stage, immediately after the introduction of the implant into the organism, the adsorption of proteins and other molecules on the surface of the material occurs. Adsorbed proteins, as well as their conformation in the native or denatured state, are specifically recognized by membrane receptors of various cell lineages. Said recognition triggers an acceptance / rejection response in the cells that determine the biocompatibility of the prosthesis.
    [0018]
    [0019] However, and even if the presence of the biomaterial does not lead to an adverse response by the organism, most prostheses are surrounded by a layer of little specialized connective tissue (connective tissue capsule). The presence of said capsule has no especially negative consequences in the short term, but it is one of the factors that limits the lifespan of many prostheses to less than twenty years. Following the biocompatibility paradigm, it is found that a possibility to extend the useful life of the prostheses and, in general, to improve the reaction of the organism to them, appears if an intimate union of the material with the surrounding functional tissue is achieved.
    [0020]
    [0021] This approach to achieve an improvement in the biocompatibility of the materials has given rise to different technologies based on the superficial modification of the prostheses. The analysis of scientific articles and patents referring to these technologies allows establishing a first classification, distinguishing between those technologies in which a polymeric material is considered or, failing that, the deposition of a polymeric coating on the material of the prosthesis and those in which the base of the coating is a thin ceramic or metal sheet. Additionally there are patent documents (see below) in which the technology developed is related to the generation of a functional layer on the coating.
    [0022]
    [0023] Within the polymeric type coatings is the production of hydrogels such as those described in patent application US2006 / 0135476 (Medical device with coating that promotes endothelial cell adherence and differentiation, 2006), in which the deposition of a polymer matrix on the substrate is the result of submerging the material into a solution that contains the polymer molecules. In this way it is possible to produce matrices of natural polymers, such as collagen or cellulose, or artificial, such as polyurethane and polyethylene glycol. Alternatively, other technologies based on the deposition of polymeric hydrogels on the surface of the materials have been proposed, such as those described in patent applications WO2006 / 105161 (Coated medical device, 2006) and WO2007 / 132099 (Method for constructing functional living materials, resulting materials and uses thereof, 2007). In the first of these patent applications, the gel is formed from natural or artificial polymers capable of adsorbing or adhering to the surface of the material, including the possibility that said polymers are chemically modified so as to contain reactive groups. In the second, hydrogels are created from polysaccharides or biodegradable peptides so that a sequential deposition of layers of these polymers with other layers containing cells occurs. A particularly unique case is the patent application WO2014 / 042463 (Synthetically designed extracellular microenvironment, 2014) in which the polymeric material is constituted by recombinant protein whose sequence is inspired by the mussel adhesive and which includes characteristic sequence motifs of proteins cell adhesion or growth factors.
    [0024]
    [0025] Alternatively to polymeric coatings, several technologies have been developed whose objective is to improve the biocompatibility of prostheses through the deposition of ceramic coatings. Hydroxyapatite deposition is one of the preferred procedures, taking advantage of the fact that hydroxyapatite is the mineral phase of the bones, which guarantees its biocompatibility. Hydroxyapatite deposition can be performed by precipitation of the mineral from a solution with the appropriate composition, as described in patent application US2015 / 0258242 (Mineralization and biological modification of biomaterial surfaces, 2015) or by an electrolytic process, such as described in patent application WO2018 / 047130 (Implantable medical devices having a coating layer with antimicrobial properties based on nanostructured hydroxyapatite, 2018). One of the main applications of these ceramic coatings is to exert an antimicrobial effect, as described in the previous patent and also in US2003 / 0077381 (Antibiotic calcium phosphate coating, 2003). Ceramic coatings have also been presented to improve biocompatibility with a composition other than Apatites including, for example, calcium aluminate, as described in patent application CA2647188 (Functionalized artificial bone and joint compositions and methods of use and manufacture, 2007) or a carbon coating, which are subsequently activated and functionalized (US2005 / 0079201; Implants with functionalized carbon surfaces, 2005).
    [0026]
    [0027] Likewise, in the state of the art various procedures have been described which comprise the union of molecules that modulate the reaction of the cells of the receptor to the coating, either polymeric or ceramic, applied on the prosthesis. In the case of polymers, technologies have been developed that allow reactive groups to be bonded to the surface of the polymer, taking advantage of the reactive groups initially present in the material, as described in US5350800 (Method for improving the biocompatibility of solid surfaces, 1993). In other cases, the binding of the molecules is preceded by a surface activation step so that they are generated in the same reactive groups, as described in patent applications WO98 / 28026 (Reactive coatings, 1998) and US20110151139 (Method for selectively functionalizing non-modified solid surface and method for immobilizing active material on the functionalized solid surface, 2011). Reactive groups created on the surface by prior activation or, in the case of polymeric biomaterials, those initially present in the implant composition are used to covalently bind various biomolecules, generally peptides or proteins, as described in EP0554869 (Biomaterials of enhanced compatibility, 1993). Within the molecules used to improve the biocompatibility of implants, it is possible to highlight, due to their originality, the use of bifunctional peptides, in which one region of the peptide has affinity for the surface to be functionalized and another region allows covalent binding to various biomolecules of interest, as described in AU2003301298 (Composition, method and use of bi-functional biomaterials, 2003).
    [0028]
    [0029] In some cases, the interaction of modified biomaterials with in vitro cell culture cells has been evaluated as a procedure to estimate the possible reaction of the organism to these materials after implantation. Thus, the patent application US2010 / 0305696 (Hybrid soft tissue implants from progenitor cells and biomaterials, 2010) presents a technology consisting of forming a hydrogel of PEGDA in which to encapsulate mammalian cells. An alternative technology is presented in US2011 / 0275539 (Substrate for selecting and specifically influencing the function of cells, 2011) in which on the surface of a biomaterial, which can be glass, are generated different domains by forming hydrogels on the surface. The objective of said hydrogels is the modulation of the functions of the cells encapsulated therein, so that the technology can be used for the in vitro analysis of the physiology or of the possible pathological conditions that affect the cells under study.
    [0030]
    [0031] As indicated above, all the strategies described have the objective of modulating the sequential reaction of certain cell lines of the recipient to the presence of the biomaterial from the moment of implantation. In order to avoid problems generally associated with currently available prostheses or implants such as, for example, their rejection by the recipient, or the formation of the non-specialized connective tissue layer (connective tissue capsule) surrounding the prosthesis or implant, what usually shortens its useful life, it would be extremely useful to achieve this modulation by means of the intervention of suitable cell lineages, intentionally introduced on the surface of the biomaterial for this purpose before implantation of the prosthesis. The achievement of this objective is not trivial, since it implies the creation of a structure that contains cells on the surface of the biomaterial which, in turn, depends on the ability to generate an adequate environment in which said cells can develop in vitro before implanting the prosthesis.
    [0032]
    [0033] On the other hand, the application of the activated steam silanization technique (SVA or its initials in English, AVS, Activated Vapor Silanization), to generate biofunctional coatings (RJ Martín-Palma et al. , Surface biofunctionalization of materials by amine groups, J. Mater. Res. 19 (2004), 2415-2420) on a substrate with a basically flat surface topography and with dimensions of the order of a square centimeter; as well as covalent immobilization of collagen on a titanium substrate (Ti-6Al-4V) previously functionalized by AVS (P. Rezvanian et al., Enhanced biological response of AVS-functionalized Ti-6Al-4V alloy through covalent immobilization of collagen, Scientific Reports 8 (2018), 3337). However, the application of the AVS technique to the functionalization of prostheses or implants with the usual dimensions of up to tens of centimeters, being able to have an area greater than 30 cm2, requires the development of new procedures and facilities not described in the state of technique
    [0034] DESCRIPTION OF THE INVENTION
    [0035]
    [0036] Next, a procedure is described that allows to create, efficiently, a prosthesis or implant with a coating (also called "second coating" or "biological coating" in this document) comprising cells adhered to a first functional coating formed by a sheet functionalized with reactive groups and, at least, an organic molecule deposited on said sheet, where this biological coating occupies a surface greater than 1 cm2, preferably greater than 30 cm2. Thus, this procedure comprises a stage where the cells are cultured in vitro, that is, prior to implantation of the prosthesis in the organism, in order to improve the patient's reaction to the prosthesis after implantation.
    [0037]
    [0038] The inventors have developed a procedure that allows the generation of a biological coating on the large surface, in particular greater than 30 cm2, of the biomaterial that forms the prosthesis, where this coating is formed prior to implanting the prosthesis in the organism. Said biological coating is generated on an appreciable fraction of the implant surface or even on the entire surface of the implant. The biological coating is formed by the combination of cells and organic molecules, in particular biomolecules, so that the cells can proliferate and differentiate on the surface of the biomaterial. The presence of this coating improves the body's response to the prosthesis.
    [0039]
    [0040] Thus, the present invention describes a procedure for obtaining a prosthesis with a coating, wherein the procedure comprises the following steps:
    [0041] a) depositing a sheet, preferably with a thickness of 20 nm to 1 ^ m, functionalized with reactive groups selected from the group consisting of amino (-NH2), carboxyl (-COOH), hydroxyl (-OH), sulfhydryl (-SH ) and a combination of the above, on at least part of the surface of a prosthesis, where deposition takes place by means of the activated steam silanization technique (SVA), and the surface where the sheet is deposited is greater than 1 cm2, preferably greater than 30 cm2;
    [0042] b) immobilize at least one organic molecule, preferably a biomolecule and / or a pharmaceutical organic compound, in the sheet deposited in step a) to obtain a first coating (also called "functional coating" in this document); and
    [0043] c) culturing at least one type of cells in vitro on the first coating obtained in step b), in particular under conditions that allow cell survival and adhesion, giving rise to a second coating (also called "biological coating" in this document ) on at least part of the surface of the prosthesis.
    [0044]
    [0045] Preferably step c) further comprises the differentiation of cultured cells to lineages compatible with the host (receptor) tissue. Thus, the procedure described in this document allows the differentiation of the cells on the functional coating, although this occurs in the environment offered by the functionalized material, an environment significantly different from the three-dimensional extracellular matrix in which the cells develop inside the recipient's organism, both from the point of view of the biomolecules that interact with the cells and from the substrate's two-dimensional geometry itself.
    [0046]
    [0047] In this document it is understood that the terms "prosthesis" and "implant" are used as synonyms. In both cases, reference is being made to the piece of material or the combination of materials to be implanted in the body and whose biocompatibility is to be improved with the process of the present invention. Thus, the prosthesis used as a substrate in the process of the present invention can be a commercial piece produced in series, or a singular piece produced on a small scale; and can be formed by various materials such as metals, ceramics or polymers.
    [0048]
    [0049] The method described in this document allows to obtain a prosthesis in whose exterior a biological coating formed by organic cells and molecules is generated, preferably biomolecules, joined to the material that forms the prosthesis (support) by means of a functionalized sheet deposited by means of SVA technology. Due to this coating, the prosthesis can be implanted in a patient in order to repair or replace damaged tissues.
    [0050]
    [0051] The main property of the prostheses of the present invention after their generation of the biological coating described herein is the improvement in their biocompatibility, as a consequence of the modification of the interaction between the organism and the prosthesis. This improvement in biocompatibility can be reflected in a decrease in implant rejection, a better interaction between the implant and the tissue surrounding and an increase in the life of the prosthesis. All these factors contribute to an improvement in the patient's general quality of life.
    [0052]
    [0053] Without being linked to any theory, the inventors consider that the improvement in the organism's response to the implant is the result of an adequate interaction between the organic tissue of the recipient and the biological phase created on the surface of the biomaterial that forms the prosthesis. In this way, the non-specific interactions established between the organic environment and the bare implant decrease. In particular, the present invention makes it possible to avoid the nonspecific adsorption of proteins and other biomolecules on the surface of the prosthesis once implanted and the cellular responses to the adsorption of said biomolecules.
    [0054]
    [0055] In preferred embodiments of the process described herein, the sheet functionalized with reactive groups is deposited on at least a part of the surface of the prosthesis with an extension greater than 1 cm2, preferably greater than 30 cm2, by a method comprising :
    [0056] ai) evaporating an organometallic compound containing at least one silicon atom and at least one functional group selected from the group consisting of amino (-NH2), carboxyl (-COOH), hydroxyl (-OH), sulfhydryl (-SH) and a combination of the above; aii) obtaining at least one flow of activated steam by heating the vapor of organometallic compound of step ai) at a temperature between 400 and 1000 ° C, preferably in an activation region with an outlet to the deposition chamber where the prostheses, so that the distance between the exit of the activation region and the surface of the prosthesis is 2 cm to 6 cm;
    [0057] aiii) having one or more activated vapor flows of step aii) affect at least a part of the surface of a prosthesis with an extension greater than 1 cm2, preferably greater than 30 cm2, where the angle of incidence of the flows of Activated vapor with respect to the surface of the prosthesis to be functionalized is 0 ° to 50 °, preferably 0 ° to 30 °, thus depositing a functionalized sheet with reactive groups selected from the group consisting of amino (-NH2), carboxyl (- COOH), hydroxyl (-OH), sulfhydryl (-SH) and a combination of the above on at least part of the surface of the prosthesis; where stages aii) and aiii) take place consecutively.
    [0058]
    [0059] The organometallic compound used in the process of the present invention is formed by molecules that have a common structure in which a silicon atom is binds to one or more hydrocarbon chains, where at least one of these chains comprises one or more amino (-NH2), hydroxyl (-OH), carboxyl (-COOH) or sulfydryl (-SH) groups. In particular, the organometallic compound comprises one or more hydrocarbon chains - (CH2) n-, where n is a number between 1 and 30, preferably between 1 and 6; and at least one functional group selected from the group consisting of hydroxyl (-OH), carboxyl (-COOH), sulfhydryl (-SH), amino (-NH2) and a combination of the foregoing. The hydrocarbon chains of the organometallic compound that is used in the process described herein can comprise one or more double or triple bonds between carbon atoms.
    [0060]
    [0061] Examples of molecules that can be used as an organometallic compound for the activated steam silanization process described herein are 3-aminopropyltriethoxysilane (APTES) and aminopropyltrimethoxysilane (APTMS), both conducive to the formation of sheets containing groups Not me; mercaptopropylmethoxysilane (MPTMS), with which sheets containing sulphydryl groups are produced; triethoxysilylpropylmalenamic acid, with which sheets containing carboxyl groups are produced; and N-triethoxysilylpropyl-O-polyethylene oxide, with which sheets containing hydroxyl groups are produced. Together with the structure of the molecule itself, an important characteristic of the organometallic is its boiling point. In particular, it is preferred that the boiling temperature of this compound be between 100 ° C and 250 ° C.
    [0062]
    [0063] The evaporation stage ai) can be carried out by depositing an organometallic fluid in an evaporation chamber located within an evaporation oven, so that the temperature of the organometallic compound can be varied. The increase in the temperature of the evaporation chamber above the boiling point of the organometallic fluid leads to the transition of liquid-vapor phase with the formation of organometallic vapor within the evaporation chamber. Preferably, the temperature range at which the evaporation oven is heated is between 50 ° C and 400 ° C, it being even more preferred that this temperature is between 100 ° C and 250 ° C and, it is especially preferred that the temperature in stage ai) of evaporation be between 130 ° C and 200 ° C. The range of evaporation temperatures will depend on the particular organometallic, with the maximum limit of the temperature of said range being the one in which the organometallic molecule decomposes.
    [0064] It is advisable to perform this evaporation stage with a pressure lower than 1 mbar, to avoid the reaction of the organometallic molecules with atmospheric gases, mainly oxygen. The need to heat the organometallic during the evaporation stage and, later, during the activation stage creates a favorable situation for the oxidation of the organometallic with atmospheric oxygen. Said reaction can decompose the organometallic, preventing the functionalization of the substrate. Thus, carrying out the method, in particular steps ai), aii) and aiii) described in this document, in a vacuum system that allows a residual vacuum between 10-4 and 10-1 mbar is advantageous to avoid decomposition mentioned above. Said vacuum can be achieved with a rotary pump coupled to a cold trap.
    [0065]
    [0066] Preferably, step aii) of obtaining at least one flow of activated steam can take place by heating the vapor of organometallic compound at a temperature between 400 and 900 ° C, more preferably between 400 and 800 ° C, since too high temperatures favor the appearance of irregularities and inhomogeneities in the deposited sheets.
    [0067]
    [0068] The stage of activation of the organometallic vapor can be carried out in an installation comprising a deposition chamber, where the prosthesis to be functionalized is located, and another region called activation region, preferably in the form of a tube, directly connected with the deposition chamber, and which has at least one outlet directed towards the deposition chamber, where this outlet is configured to allow the flow of activated steam to influence the surface of the prosthesis to be functionalized at an angle between 0 ° and 50 °, preferably between 0 ° and 30 °, and a distance between the output of the activation region and the prosthesis preferably 2 cm to 6 cm. According to particular embodiments of this installation, the activation region corresponds to the region of the tube immediately before the deposition chamber and which connects directly with it. Additionally, the installation comprises an activation furnace located around the activation region, defining its extension and allowing a controlled temperature increase in said activation region. Thus, in the activation region, the evaporated organometallic vapor in the evaporation chamber crosses a high temperature region, before entering the deposition chamber and influencing the surface of the prosthesis to be functionalized with the appropriate angle to perform an efficient functionalization of at least a part of the surface of a prosthesis with an extension greater than 1 cm2, preferably with an extension greater than 30 cm2.
    [0069] The application of the SVA technique to the functionalization of prostheses, with dimensions generally greater than 1 cm2, requires the development of specific procedures and facilities so that the deposition of the sheet on the surface of the prosthesis to be functionalized takes place in a homogeneous way and efficient.
    [0070]
    [0071] Thus, in especially preferred embodiments of the present invention, the functionalization of areas greater than 1 cm2 and, in particular, greater than 30 cm2 can be achieved by the procedure described herein, where step aiii) takes place in a installation comprising a deposition chamber adapted to the size and geometry of the prosthesis to be functionalized, so that the activated steam flows are introduced into the deposition chamber through different inputs, each of these inputs connected to the output of a independent activation region, where the outputs of the different activation regions are configured such that the flow of activated steam affects the surface of the prosthesis to be coated with an angle between 0 ° and 50 °, preferably between 0 ° and 30 ° , and where the distance between the output of each of these activation regions and the surface of the prosthesis remains fixed at an e-value. established between 2 cm and 6 cm (see figure 2a).
    [0072]
    [0073] Alternatively, step aiii) of the procedure described herein may take place in an installation such as that described in the previous paragraph, with the proviso that the distance between the output of the different activation regions and the surface of the prosthesis to be functionalized is you can modify during the procedure, for example, using flexible vacuum connectors as outputs of the activation regions (see figure 2b). In this case, the deposition chamber has a variable geometry with respect to the distance between the activated steam outlet and the surface of the sample to be functionalized, so that it is possible to work with different geometries and sizes of prostheses in a single camera.
    [0074]
    [0075] Preferably, in step aiii) of the procedure described herein, the period of incidence of the activated organometallic vapor flows on at least part of the surface of the prosthesis is between 1 and 120 minutes, more preferably between 5 min and 30 min.
    [0076]
    [0077] Without being linked to any theory, it is believed that the functionalization of the prosthesis in particular embodiments of the present invention is the result of the activation of organometallic vapor, and the subsequent incidence of the molecules thus activated in the part of the surface of the prosthesis to be functionalized, resulting in the formation of a sheet, preferably with a thickness of 20 nm to 1 µm, more preferably 20 nm to 300 nm. A scheme of the installation or equipment that can be used to carry out the functionalization process of the prosthesis by activated steam silanization with an activated steam line is shown in Figure 1 (a), schematically indicating the result of the process of functionalization in Figure 1 (b). Additionally, figures 2 (a) and 2 (b) show diagrams of suitable equipment for use in those embodiments of the process of the invention, where step aiii) comprises influencing independent activated steam flows on different areas of the prosthesis .
    [0078]
    [0079] Although in the procedure described herein it is preferred to deposit the functionalized sheet on the entire external surface of the prosthesis. It is also possible to deposit a functionalized sheet with a reactive group selected from the group consisting of amino (-NH2), carboxyl (-COOH), hydroxyl (-OH), sulfhydryl (-SH) and a combination of the above only in part of The surface of the prosthesis. For this, a mask can be used, for example, of photolithography, which allows dividing the surface of the prosthesis into different sections, so that only a part of the surface of the prosthesis can be functionalized or, alternatively, deposit sheets with different characteristics and, in particular, with a differentiated functionality in different sections of the surface of the prosthesis.
    [0080]
    [0081] As mentioned above, step b) of the process described herein comprises immobilizing at least one organic molecule in the sheet deposited in step a), preferably this sheet has a thickness of 20 nm to 1 pm and, more preferably between 20 nm and 300 nm. For this, the reactive groups present in the deposited sheet are reacted with at least one organic molecule capable of forming bonds with these reactive groups. In this way a suitable niche is generated that favors proliferation and, preferably, cell differentiation, in at least part of the surface of the prosthesis.
    [0082]
    [0083] Preferably, the organic molecules incorporated into the prosthesis coating in step b) of the process described herein react with the reactive groups of the functionalized sheet by means of covalent bonds, or other types of bonds that have comparable stability. In particular, the stability of the bond between biomolecules and / or organic molecules with the surface of the prosthesis is optimal by means of establishment of a covalent bond with the reactive groups of the functional sheet deposited in step a).
    [0084]
    [0085] The procedure described herein allows immobilizing different organic molecules, in particular pharmaceutical active ingredients and / or biomolecules, on the surface of the prosthesis where the functional sheet has been deposited. Thus, for example, the surface of the prosthesis, functionalized by SVA as described herein, can be divided into different sections, in particular sections with a size of 1 mm2 to 10 cm2, preferably 1 cm2 to 10 cm2, in order to immobilize different organic molecules in these sections.
    [0086]
    [0087] In this patent application it should be understood that "pharmaceutical active ingredient" (also called "pharmaceutical organic compound" in this document) refers to a chemical substance with pharmacological effect.
    [0088]
    [0089] The presence of reactive groups on the surface of the prosthesis allows direct covalent bonding of biomolecules and / or other organic molecules of smaller size, in particular pharmaceutical active compounds, that is, chemical substances with pharmacological effect, such as, for example, antibiotics, with the material of the prosthesis through the reactive groups present in them (hydroxyl (-OH), sulfydryl (-SH), carboxyl (-COOH) and / or amino (-NH2)). The improvement in biocompatibility associated with the presence of these organic molecules on the surface of the prosthesis depends on the functions that these molecules develop within the body. In particular, the following functions can be set:
    [0090]
    [0091] - Infection prevention. The immobilization of antibiotic molecules has as an additional function the elimination of possible pathogens present on the surface of the prosthesis, thereby reducing the risk of rejection of the coated prosthesis object of this invention.
    [0092]
    [0093] - Modulation of cell adhesion. The choice of the immobilized molecule in the functionalized sheet can allow modulating cell adhesion, through the specific interaction of the selected molecules and the membrane receptors present in the cells. This modulation can be aimed at improving the adhesion of functional cells, or with the ability to differentiate functional cells, that is, from the cells grown in vitro in step c) of the procedure described herein. An example of this type of modulation is the immobilization of extracellular matrix proteins, such as collagen, fibronectin or laminin. For this purpose, the complete molecule can be immobilized, or only one or more of the elements or fragments that compose it. As an example of this second case there is the reason for the RGD (Arginine-Glycine-Aspartic) sequence characteristic of the fibronectin sequence and which is recognized by various membrane receptors in the cells. Among the proteins of the extracellular matrix, fibronectin immobilization is preferred, since it provides optimal results in terms of the total number of cells on the surface and their size.
    [0094]
    [0095] - Alternatively, modulation can result in a decrease in cell adhesion, the most extreme example being found in the production of surfaces with low bacterial adhesion. In particular, this objective can be obtained by immobilizing albumin in the functional sheet deposited on the surface of the prosthesis.
    [0096]
    [0097] - Improvement of survival, growth and, eventually, cell differentiation. Within the group of molecules that can be immobilized to obtain an improvement in survival, growth and, eventually, cell differentiation in the coated prosthesis of the present invention, there are cellular signaling molecules such as growth factors, in particular, the factor of epithelial growth (EGF), neuronal growth factor (NGF) or vascular endothelial growth factor (VGEF); as well as cytokines, anti-inflammatory and angio-gene biomolecules, and other soluble molecules that mediate cell signaling through various receptors. Thus, these molecules can be found both in the culture medium used in step c), and immobilized in the functionalized sheet deposited on the surface of the prosthesis. In this second case, the molecules bind to the functionalized sheet preferably through biodegradable crosslinking agents, so that the release of the biomolecules to the medium can be performed in a controlled manner and with known kinetics. As an example of biodegradable crosslinking agents are protease sensitive peptides, such as the Pro-Val-Gly-Leu-Ile-Gly and Pro-Leu-Gly-Leu-Ala-Gly hexapeptides. In both cases, metalloproteases II (MMP-2) and 9 (MMP-9) induce the specific breakage of the peptide bond between the glycine and leucine residues.
    [0098]
    [0099] Although the presence of reactive functional groups both in the sheet deposited on the surface of the material and in the molecules to be immobilized in this sheet can allow the creation of a direct covalent bond between both elements, the formation of said covalent bond is more efficient by the use of at least one crosslinking agent.
    [0100]
    [0101] Crosslinking agents are organic molecules that typically contain two functional groups and a spacer region. The crosslinking agents favor the reaction between the reactive groups of the functionalized sheet and the reactive groups of the biomolecules and / or organic molecules that it is desired to immobilize on this sheet. If the crosslinking agent itself does not act as a link between the sheet and the molecule, it is called of zero length. Frequently the crosslinking agent constitutes a stable part of the system together with the functionalized sheet and the organic molecules to be immobilized by allowing covalent bonding between both elements. As an example of a null length crosslinking agent is EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) which allows the creation of a covalent bond (peptide bond) between an amino group (-NH2) and a group carboxyl (-COOH). As an example of a crosslinking agent of non-zero length is glutaraldehyde, which allows the covalent attachment of two amino groups. For some applications, crosslinking agents that undergo spontaneous or dependent degradation of environmental conditions may be used, so as to allow the release of biomolecules or organic molecules initially covalently attached to the functionalized sheet deposited in the prosthesis. This last aspect is very interesting for the controlled release of drugs and different biomolecules, being able to alternate regions of the surface of the prosthesis where biodegradable crosslinking agents are used, for example, to release growth factors that favor the development of cells on the surface of the prosthesis, and others in which the bonds are stable, for example, to bind biomolecules that modulate cell adhesion.
    [0102]
    [0103] When the molecule to be immobilized is a biomolecule and, in particular, a protein, in step b) of the process described herein, the use of the system formed by EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) is preferred. and NHS (N-hydroxysuccinimide) as crisscross These compounds are not integrated into the structure formed by the functionalized sheet and the biomolecule and, therefore, this system is considered of zero length. An example of the result of using the EDC / NHS system as crosslinking agents to covalently bind a protein to a biofunctionalized surface is presented in Figure 3.
    [0104]
    [0105] In particular embodiments of the present invention, the process described herein may comprise a step of cleaning the surface of the prosthesis with the functional coating, that is, after step b) of the process. The inclusion of this cleaning step makes it possible to eliminate traces of crosslinking agent that can give rise to a toxic effect on the cells. In particular, when the system formed by EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide) is used as a crosslinker, this cleaning step comprises several sub-stages of incubation of the functionalized prosthesis with at least one immobilized organic molecule (prostheses with a first functional coating) with different solutions, in particular, PBS buffer (phosphate buffered saline), MES buffer (ie 2- (N-morphorelin) ethanesulfonic acid buffer) and / or DMEM (known in English as Dulbecco's Modified Eagle Medium).
    [0106]
    [0107] In particularly preferred embodiments, the cleaning step is carried out following the procedure detailed below:
    [0108] bi) cleaning the prosthesis with the first coating with distilled water, preferably immediately after removing the solution of crosslinking agent;
    [0109] bii) incubate the prosthesis with PBS buffer (Na2HPO410 mM, KH2PO41.8 mM, 137 mM NaCl, 27 mM KCl; pH = 7.4) for a period between one and four, preferably for 2 hours; where this incubation step is performed n times, where n is a value between 1 and 4, preferably n is equal to 2;
    [0110] biii) incubate the prosthesis obtained after step bii) with MES buffer (0.2 M; pH = 6.0) for a period between 24 h and 96 h, preferably for 72 hours; where this incubation step is performed l times, with l being a value between 1 and 4, preferably l is equal to 2;
    [0111] biv) incubate the prosthesis in DMEM (1x) for a period between 6 and 24 hours, preferably for 12 hours; where this incubation step is performed m times, m being a value between 1 and 4, preferably m is equal to 2.
    [0112] An alternative procedure for the stable binding of biomolecules to the sheet deposited on the surface of the prosthesis is offered by the interaction in the streptavidin-biotin system (or, alternatively, avidin-biotin). The streptavidin-biotin junction, despite not being properly a covalent bond, turns out to be of comparable intensity. In this context, it is possible to bind streptividin molecules covalently to the functional groups of the lamina deposited on the surface of the prosthesis, joining in a process that develops in parallel, biotin molecules to the biomolecules that are desired to immobilize on the functionalized sheet (biotinylation of biomolecules). Incubation of said functionalized sheet with streptavidin immobilized covalently with the biotinylated biomolecules medium causes immobilization of said biomolecules on the sheet deposited on the surface of the prosthesis.
    [0113]
    [0114] The modifications made on at least part of the surface of the prosthesis in stages a) and b) are aimed at creating a niche that allows survival, growth and, eventually, the differentiation of the desired cell lines in vitro before proceeding to implant the prosthesis in the organism, as well as improve their survival after the introduction of the prosthesis in the organism.
    [0115]
    [0116] In a similar way to that indicated in relation to the previous stages, the procedure described in this document allows to cultivate different cell lineages in different sections of the surface of the prosthesis.
    [0117]
    [0118] The choice of cell lineage will depend, in each case, on the type and function of the prosthesis and, in particular, on the type of tissue that will come into direct contact with the prosthesis after implantation. In turn, the choice of cell lineage and the development that is induced on the surface of the prosthesis (survival, growth and / or differentiation) determines the biomolecules and / or organic molecules immobilized on the surface functionalized in step b) of the procedure . As mentioned above, these molecules allow modulating the behavior of the cells on the surface of the prosthesis. Additionally, cell culture of the cells will involve the addition of a culture medium with a specific composition depending on the type of development that is desired in the cells.
    [0119] Although there are in principle no restrictions on cell lineages that can be used in the context of the present invention, it is preferred to use pluri- and multipotent cell lineages, since they offer the potential to create specialized tissue on the prosthesis.
    [0120]
    [0121] In particular, the use of multi-potent stem cells such as, for example, mesenchymal cells from bone marrow (BM-MSC) and osteochondroreticular lineage (OCR) cells is preferred. These cells can fulfill a series of complementary functions, among which are the limitation of the inflammatory process associated with the introduction of the implant into the organism, the promotion of the creation of healthy tissue in the environment of the prosthesis and, even, the direct differentiation from multipotent cells cultured on the surface to different tissues, such as bone, cartilaginous, adipose or muscular tissues.
    [0122]
    [0123] As a source of the cells it is preferable to use the patient's own cells (autologous origin), since this procedure considerably reduces the possibility of rejection. Alternatively, cells from a human donor (allotransplant) can be used. However, in the case of mesenchymal stem cells, their immunosuppressive nature and limited immunogenicity facilitates the implantation of prostheses in an autologous and allogeneic way.
    [0124]
    [0125] A particularly advantageous aspect of the procedure described in this document is that it allows not only the growth of the various cells, but also differentiates them into various cell lines, an aspect based on the ability of multi-potent and multi-cell cells. powerful to generate virtually any cell population. Thus, the prosthesis with the biological coating obtained by this procedure offers the possibility of growing natural pluri-potent cells, such as induced pluri-potent stem cells that can be obtained by reprogramming based on integrative methods (with recombinant DNA) or non-integrative (RNA, recombinant proteins or simple incubation with factors). It is also possible to grow multi-potent stem cells from the mesoderm, endoderm, ectoderm lineages; and achieve, as with pluripotent stem cells, their differentiation to different populations of differentiated cells.
    [0126]
    [0127] Additionally, the present invention allows to directly reprogram already differentiated cells, in such a way that other cell lines are generated on the biological coated prosthesis. For example, to generate nerve tissue it is possible to grow Human fibroblasts on the functionalized prosthesis with a coating comprising a functionalized sheet and organic molecules, in particular biomolecules, immobilized on said sheet. These fibroblasts can be transduced with certain factors to generate glutamatergic and GABAergic neurons (ASCL1, BRN2, MYT1L factors), dopaminergic neurons (ASCL1, NURR1, LMX1A), mature astrocytes (NFIA, NFIB, and SOX9 factors), oligondendrocytes or myelin-forming cells (sox10, Olig2, Nkx6-2 factors) and endothelial cells for blood vessel production (ER71 / ETV2 factors).
    [0128]
    [0129] Additionally, the use of multi-potent stem cells of mesenchymal origin from bone marrow (BM-MSC) allows the cells to differentiate themselves to several cell lines on the prosthesis obtained by the procedure described herein. In particular, for bone prostheses it is convenient to differentiate BM-MSC cells to osteoblasts, while for joint prostheses it is convenient to differentiate said cells to chondrocytes, as well as the differentiation of BM-MSC cells to muscle cells.
    [0130]
    [0131] Another aspect of the present invention is the coated prosthesis (also referred to as a biological coated prosthesis in this document) obtained by the procedure described herein.
    [0132]
    [0133] In preferred embodiments, this coated prosthesis comprises:
    [0134] - a prosthesis with at least one surface of biocompatible material, preferably metallic, ceramic or polymeric;
    [0135] - a first coating (also called "functional coating") comprising:
    [0136] or a sheet functionalized with reactive groups selected from the group consisting of amino (-NH2), carboxyl (-COOH), hydroxyl (-OH), sulfhydryl (-SH) and a combination of the above, preferably this sheet has a thickness between 20 nm and 1 ^ m, more preferably between 20 nm and 300 nm, deposited on at least a part of the surface of the prosthesis with an extension greater than 1 cm2, more preferably greater than 30 cm2; Y
    [0137] or at least one organic molecule, preferably fibronectin, immobilized in the functionalized sheet; and
    [0138] - a second coating (also called "biological coating") comprising multi-potent or multi-potent cells adhered to the first coating.
    [0139]
    [0140] In particular, the cells adhered to the functional coating are preferably BM-MSC mesenchymal cells that have the ability to differentiate into specialized lineages such as osteoblasts and chondrocytes. These types of prostheses are particularly advantageous for use as prostheses in trauma applications.
    [0141]
    [0142] In preferred embodiments, the differentiation of BM-MSC cells to chondrocytes can take place using a DMEM culture medium supplemented with 5 to 10% fetal bovine serum (SFB) and enriched with a series of supplements that may be a basal insulin medium. Transferrin-selenium, sodium pyruvate, dexamethasone, ascorbic acid, transforming growth factors, L-Glutamine, and antibiotics such as penicillin or / and streptomycin.
    [0143]
    [0144] Brief description of the figures
    [0145]
    [0146] The figures included in the patent application document are:
    [0147]
    [0148] Figure 1a: Scheme of the device (also called “installation” in this document) that can be used for stage a) of activated steam silanization (SVA) with a single activated steam flow, where the main constituent elements of this device:
    [0149] - An evaporation chamber (3), where the organometallic compound (2) is located during the process of the invention. This chamber is at least partially surrounded by an evaporation oven (4), has an inlet (8) of carrier gas (for example, argon) and a steam outlet (9) of organometallic compound (9).
    [0150] - An activation region (6a) connected to the evaporation chamber (3) so that the vapor of organometallic compound can circulate from said evaporation chamber (3) to the activation region (6a), an outlet (10a) of the flow of activated steam generated in this region during the process of the invention, and an activation furnace (5a) that at least partially covers the activation region (6a). In the preferred case shown in Figure 1a, the activation region (6a) is a tube through which steam of organometallic compound circulates, resulting in the flow of activated steam at the outlet (10a) of the activation region.
    [0151] - A deposition chamber (7a), where the prosthesis (1) to be coated is located, connected to the outlet (10a) of the activation region so that the angle of incidence (a) of the vapor flow activated in the The surface of the prosthesis to be coated is between 0 ° and 50 °, and the distance (d) between the outlet (10a) and the surface of the prosthesis to be coated is preferably between 2 cm and 6 cm.
    [0152] - Optionally, the deposition chamber (7a) can be connected to a vacuum pump (11), as shown in Figure 1a.
    [0153] - Additionally, the deposition chamber (7a) can comprise a motorized sample holder (not shown in the figure), to modify the orientation of the prosthesis (1) with respect to the only steam flow inlet activated from the outlet (10a) of the activation region.
    [0154]
    [0155] Figure 1b: Extended diagram of the outlet (10a) of the activation region (6a) to the deposition chamber (7a) containing the prosthesis (1) to be coated, where the angle of incidence (a) of the flow is represented of activated steam on the surface of the prosthesis (1) to be coated is between 0 ° and 50 °, and the distance (d) between the outlet (10a) and the surface of the prosthesis to be coated, preferably between 2 cm and 6 cm.
    [0156]
    [0157] Figure 1c: Scheme of the result of an activated steam silanization process, using 3-aminopropyltriethoxysilane (APTES) as an organometallic fluid, resulting in the deposition of a thin sheet with amino groups (NH2) on the surface.
    [0158]
    [0159] Figure 2a: Scheme of part of the device that can be used for step a) of activated steam silanization (SVA), when several independent flows of activated steam strike the prosthesis. As shown in this figure, this device is similar to that shown in Figure 1a, with the proviso that the deposition chamber (7b) is connected to more than one activation region (6b), each of these regions has a output (10b) of the flow of activated steam generated during the process of the invention, and an activation furnace (5b) that at least partially covers the activation region (6b). Although not shown in this figure, each of these activation regions (6b) can be connected to it or to different evaporation chambers, so that the vapor of organometallic compound can circulate from the aforementioned chamber (s) (s) of evaporation to the activation regions (6b) and, subsequently, to the deposition chamber (7b), In the preferred case shown in Figure 2a, the device consists of 5 activation regions (6b), all of them in tube form where compound vapor circulates organometallic, giving rise to different flows of activated steam to the outlets (10b) of the activation regions. Additionally, the deposition chamber (7b) may comprise a motorized sample holder (not shown in the figure), to modify the orientation of the prosthesis (1) with respect to the steam flow inputs activated from the outlet (10b) of the regions of activation.
    [0160]
    [0161] Similarly to the previous embodiment, the deposition chamber (7b), where the prosthesis (1) to be coated is located, is connected to the outputs (10b) of the activation regions so that the angle of incidence (a ) of the flow of activated steam on the surface of the prosthesis to be coated is between 0 ° and 50 °, and the distance (d) between each of the outlets (10b) and the surface of the prosthesis to be coated remains fixed for the procedure in a value, preferably, between 2 cm and 6 cm.
    [0162]
    [0163] Figure 2b: Scheme of part of the device that can be used for step a) of activated steam silanization (SVA), when several independent flows of activated steam strike the prosthesis. The system shown in this figure is the same represented in Figure 2a, with the proviso that the outputs (10c) of the activation regions (6c) are flexible, in particular, flexible vacuum connections, so that distance ( d) between each of the outlets (10c) and the surface of the prosthesis to be coated (1) it can be modified during the procedure in a value, preferably, keeping it at all times between 2 cm and 6 cm. This configuration allows the use of a single activation chamber to functionalize different prostheses in terms of their geometry and size.
    [0164]
    [0165] Figure 3: Schematic representation of the covalent bond that is established between the -NH2 groups of a functionalized sheet with the -COOH groups of a protein (indicated by P in the Figure) by using 1-ethyl-3- (3- dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as crosslinking agents.
    [0166]
    [0167] Figure 4: Result of applying the different stages of the procedure to a sample of Ti6Al4V. (A) Atomic force microscopy image of the Ti6Al4V control sample. (B) Atomic force image of a sample not functionalized by SVA, incubated with EDC / NHS and fibronectin and subsequently washed with an aqueous detergent solution (SDS). (C) Atomic force microscopy image of a sample functionalized by SVA, incubated with EDH / NHS and fibronectin and subsequently subjected to washing with an aqueous detergent solution (SDS). The presence of the protein on the surface after the washing process. (D) Image of a cell culture of mesenchymal stem cells from bone marrow (BM-MSC) on a surface functionalized by SVA and to which fibronectin has been covalently bound. The image has been taken with a fluorescence microscope after staining the cells with the calcein vital dye denoting the viability of the cell culture on the functionalized surface.
    [0168]
    [0169] Figure 5: Growth of BM-MSC cells on Ti6Al4V after 48 hours of culture observed by fluorescence microscopy using the phallus / Hoechst marker. (A) Sample of Ti6Al4V control. (B) Sample of Ti6Al4V functionalized by SVA to which fibronectin has been covalently bound, using EDC / NHS as crosslinking agents.
    [0170]
    [0171] Figure 6: Effect of fibronectin coating on the adhesion of BM-MSC cells on Ti6Al4V. BM-MSC cells were cultured on control (plastic) (figures 6A (0 min) and 6B (1 min)), Ti6Al4V not functionalized (figures 6C (0 min) and 6D (1 min)) and Ti6Al4V functionalized with fibronectin following the procedure described below (figures 6E (0 min) and 6F (1 min)). The samples were treated with trypsin for the indicated time (0 min or 1 min) and the number of cells on each of the materials was compared. It is observed that after incubation with trypsin for one minute there are no cells left on the surface of the non-functionalized Ti6Al4V (Figure 6D), in contrast to the maintenance of an appreciable number of cells both in the control and in the Ti6Al4V functionalized with fibronectin ( figure 6F).
    [0172]
    [0173] Figure 7: Differentiation of BM-MSC cells to chondrocytes on the surface of Ti6Al4V functionalized with fibronectin. BM-MSC grown on plastic (control) (Figures 7A and 7B) and on Ti6Al4V functionalized with fibronectin (Figures 7C and 7D) are compared. The cells are observed after 10 days of culture in proliferation medium similar to that used in the cell cultures of Figures 5 and 6 (Figures 7A and 7C) or in differentiation medium (Figures 7B and 7D). Hoescht staining has been used to identify cells (blue) and a monoclonal anti-aggrecan antibody to identify aggrecan deposition, revealed with a mouse anti-mouse IgG fluorescent antibody (red), as characteristic activity of chondrocytes. A higher density of aggrecan deposits is observed on the Ti6Al4V sample functionalized with fibronectin (Figure 7D) compared to the control (Figure 7B).
    [0174] Examples
    [0175]
    [0176] The following examples are included to provide experts in the area with a complete description of how to make and apply the present invention. They should not be considered in any way to limit the scope of what the inventors consider as their invention, nor should they be supposed to constitute a complete enumeration of all the experiments performed.
    [0177]
    [0178] Examples will be presented that illustrate each of the steps that comprise the fabrication of the bi-coated prostheses object of this invention. Consequently, it should be understood that the fabrication of this prosthesis requires the sequential realization of one of the examples proposed for each of the stages.
    [0179]
    [0180] Example 1.1: Deposition of a functionalized sheet on the surface of the prosthesis by activated steam silanization with a single line of activated steam flow.
    [0181]
    [0182] The following Table shows the range of preferred parameters of the functionalization process by activated steam silanization compatible with the deposition of functionalized sheets, in particular with a thickness of 20 nm to 300 nm on Ti6Al4V alloy prostheses.
    [0183]
    [0184]
    [0185]
    [0186]
    [0187]
    [0188] In particular, it is found that with the following set of process parameters (T evaporation = 150 ° C; T activation = 750 ° C; p = 1 mbar; t deposition = 20 min) a functionalized sheet with a thickness of approximately 100 nm Alternatively, the use of the same conditions, but using an argon pressure of p = 2 mbar leads to the deposition of a functionalized sheet of 200 nm. In both cases, a surface density of surface amino groups estimated in 8 -NH 2 groups per nm 2 is obtained , close to the theoretical value of a monolayer of amino groups on a surface.
    [0189]
    [0190] Example 1.2: Covalent binding of biomolecules or organic molecules to the functionalized sheet reactive groups.
    [0191]
    [0192] 1.2.1. Fibronectin immobilization
    [0193]
    [0194] Due to its natural abundance in the organism and its key function as a component of the extracellular matrix, fibronectin is one of the most widely used molecules in order to modulate the body's response to the prosthetic implant. Below is a procedure with which it is possible to covalently immobilize fibronectin on a surface of a prosthesis previously functionalized by SVA (in particular, when the functionalization takes place as described in example 1.1 of this document). This immobilization procedure is based on the use of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as crosslinking agents. As these crosslinking agents are not integrated into the structure formed by the functionalized sheet and the protein, this system is considered of zero length. An example of the result of using the EDC / NHS system as crosslinking agents to covalently bind a protein to a biofunctionalized surface is depicted in Figure 3.
    [0195]
    [0196] More specifically, for covalent immobilization of fibronectin on a functionalized surface with a high surface density of -NH 2 groups, a solution of fibronectin with a concentration of 200 ^ g / ml of fibronectin in 0.1 M buffer is used. MONTH (2- (N-morpholin) ethanesulfonic acid), the prosthesis being incubated for one hour in said solution. Subsequently an EDC / NHS solution in buffer is added MONTH so that the final concentration of both crosslinking agents in the solution is 0.125 mg / ml (EDC) and 0.0315 mg / ml (NHS). The prosthesis is incubated for four hours in this solution, finally cleaning (see below) to eliminate possible remains of free EDC and NHS that have not participated in the reaction.
    [0197]
    [0198] The cleaning of the sample took place as indicated below. Immediately after removing the crosslinker solution, the sample was washed with distilled water and, subsequently, two incubation processes were performed in PBS buffer (2 hours each incubation). The incubation of the treated sample in MES buffer was then carried out for a period of 72 hours and, finally, two cycles of incubation were performed in DMEM (12 hours each incubation).
    [0199]
    [0200] 1.2.2. Albumin immobilization
    [0201]
    [0202] In a second variant, the protocol described in section 1.2.1 can be modified to immobilize albumin. To this end, a solution of 5 mg / ml of bovine serum albumin (BSA) in MES buffer at a concentration of 0.1M is incubated by incubating with a previously functionalized prosthesis (in particular, following the procedure deposition described in example 1.1 of this document) for 1 hour. After this time, EDC / NHS is added as crosslinking agents, so that the final concentrations of each of the components are: BSA-2.5 mg / ml, EDC-2.5 mg / ml, NHS-0.63 mg / ml Incubation of the prosthesis is maintained for 4 hours, washing with distilled water to remove excess BSA not covalently bound to the material.
    [0203]
    [0204] Example 1.3: Cell culture on the modified prosthesis by the presence of the functional sheet and the immobilized molecules in said sheet in a proliferation culture medium
    [0205]
    [0206] Multi-potent stem cells of mesenchymal origin from bone marrow (BM-MSC) can be obtained by biopsy of the donor's long bones, which, if their general condition allows, can be the posterior recipient of the prosthesis with the biological coating (autotransplantation).
    [0207] From the biopsy it is possible to isolate and expand the MSC cells following the protocol detailed below. First, the cellular content obtained after the biopsy is filtered and centrifuged, recovering the cell-rich sediment. The cells thus obtained are cultured in complete medium for human mesenchymal cells called MesenCult, containing basal medium supplemented with specific factors for human cells (Mesenchymal Stem Cell Stimulatory Supplements and MesenCult® MSC Basal Medium, Stem Cell Technologies Human; Catalog # 05402 and Catalog # 05401), as well as 100 U / mL penicillin and 100 pg / mL streptomycin. Once at least five culture passes have been made, with the consequent enrichment in mesenchymal cells, the following passes are made in a supplemented MesenCult medium 100 U / mL penicillin and 100 pg / mL streptomycin. The use of cells with a number of passes between 5 and 15 is preferred for use in biological coated prostheses.
    [0208]
    [0209] BM-MSC cells can be cultured on the previously sterilized prosthesis. Sterilization of the prosthesis is performed by exposure to ultraviolet radiation so that the entire surface of the prosthesis is exposed for a minimum of 20 minutes to the radiation. The cells are deposited at a concentration of 2.5 * 104 cells per cm2 of prosthesis, the culture being maintained in the same medium used between passes 5 and 15, this is MesenCult supplemented medium 100 U / mL penicillin and 100 pg / mL streptomycin .
    [0210]
    [0211] Figure 4 illustrates the result of the three stages of which the procedure consists following the example described. Figure 5 compares the survival and cell growth of BM-MSC samples on a control sample of Ti6Al4V (Figure 5A) with a sample functionalized by SVA to which fibronectin has been covalently linked. Figure 6 shows the improvement in adhesion of BM-MSC cells on the surface of the Ti6Al4V functionalized with fibronectin (Figure 6F) compared to the non-functionalized material (Figure 6D). In this case, both samples have been subjected to an incubation with trypsin for 1 minute. No cells are observed on the surface of the non-functionalized material after treatment, in contrast to the presence of a large group of cells in the functionalized material.
    [0212]
    [0213] Example 1.4: Differentiation of cells on the modified prosthesis by the presence of the functional sheet and the immobilized molecules in said sheet in differentiation culture medium
    [0214] The differentiation of BM-MSC cells to chondrocytes has been achieved using a culture medium with the following composition: DMEM with high glucose (Gibco) supplemented with 10% fetal bovine serum (SFB), 1X basal insulinatransferrin-selenium medium ( Gibco, with a final concentration of 10mg / l of bovine insulin, 5.5 mg / l of transferrin, 5 ^ g / l of sodium selenium, 4.7 ^ g / l of linoleic acid and 9.5 mg / ml of bovine serum albumin), 1 mM sodium pyruvate, 0.1 ^ M dexamethasone, 50 ^ g / ml ascorbic acid, 10 ng / ml transforming growth factor (TGF-Beta1, Preprotech), 100 U / ml penicillin and 100 ^ g / ml streptomycin and 2 mM L-glutamine. The differentiation has been analyzed after 10 days of culture in differentiation medium, through the use of immunofluorescence with an anti-aggrecan antibody, since this is a characteristic chondrocyte polysaccharide. Figure 7 shows how BM-MSC cells differentiated in the Ti6Al4V substrate functionalized with fibronectin (Figure 7D) have a secretion of aggrecan much higher than the cells grown on the control (Figure 7B).
    权利要求:
    Claims (16)
    [1]
    1. - A procedure for obtaining a prosthesis with a coating, characterized in that the procedure comprises the following steps:
    a) depositing a functionalized sheet with reactive groups selected from the group consisting of amino (-NH2), carboxyl (-COOH), hydroxyl (-OH), sulfhydryl (-SH) and a combination of the above, on at least part of the surface of a prosthesis, where deposition takes place by means of the activated steam silanization technique and the surface where the sheet is deposited is greater than 1 cm2;
    b) immobilize at least one organic molecule in the sheet deposited in step a) to obtain a first coating; Y
    c) culturing at least one type of cells in vitro on the first coating obtained in step b), resulting in a second coating on at least part of the surface of the prosthesis.
    [2]
    2. - The method for obtaining a prosthesis according to claim 1, wherein step c) further comprises differentiation of cultured cells to lineages compatible with the host tissue.
    [3]
    3. - The method for obtaining a prosthesis according to any one of claims 1 to 2, wherein the prosthesis is formed by a material selected from the group consisting of metallic, ceramic and polymeric.
    [4]
    4. - The method for obtaining a prosthesis according to any one of claims 1 to 3, wherein step a) comprises the following sub-stages:
    ai) evaporating an organometallic compound containing at least one silicon atom and at least one reactive group selected from the group consisting of amino (-NH2), carboxyl (-COOH), hydroxyl (-OH), sulfhydryl (-SH) and a combination of the above;
    aii) obtaining at least one flow of activated steam by heating the vapor of organometallic compound of step ai) at a temperature between 400 and 1000 ° C;
    aiii) having one or more activated vapor flows of step aii) affect at least a part of the surface of a prosthesis with an extension greater than 1 cm2, where the angle of incidence of activated vapor flows with respect to the surface of the prosthesis to be functionalized is 0 ° to 50 °;
    where stages aii) and aiii) take place consecutively.
    [5]
    5. - The method for obtaining a prosthesis according to claim 4, wherein step aiii) comprises causing independent activated vapor flows to influence different areas of the surface of the prosthesis.
    [6]
    6. - The method for obtaining a prosthesis according to any one of claims 4 to 5, wherein step aiii) takes place in a deposition chamber (7) connected at least to an activation region (6a, 6b, 6c), each of these regions with an activated steam flow outlet (10a, 10b, 10c) located at a distance between 2cm and 6cm from the surface of the prosthesis to be functionalized.
    [7]
    7- The method for obtaining a prosthesis according to any one of claims 1 to 6, wherein step b) comprises immobilizing an organic molecule selected from the group consisting of biomolecule, pharmaceutical active compound and a combination of the foregoing.
    [8]
    8. - The method of obtaining a prosthesis according to claim 7, wherein
    - the biomolecule is selected from the group consisting of extracellular matrix proteins, albumin, cell signaling molecules, a fragment of any of these biomolecules and a combination of the above; or
    - the pharmaceutical active compound is an antibiotic.
    [9]
    9. - The method of obtaining a prosthesis according to claim 8, wherein the extracellular matrix protein is fibronectin or fragments of said protein.
    [10]
    10. - The method of obtaining a prosthesis according to any one of claims 1 to 9 wherein step b) takes place in the presence of at least one crosslinking agent.
    [11]
    11. - The method of obtaining a prosthesis according to claim 10, wherein the Organic molecule is a biomolecule, and the crosslinking agent is a combination formed by EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide).
    [12]
    12. - The method for obtaining a prosthesis according to any one of claims 1 to 11, wherein step c) comprises culturing multi-potent or multi-potent cells.
    [13]
    13. - A coated prosthesis, characterized in that the coating comprises a first coating with cells adhered to this coating, wherein this coated prosthesis is obtained by the method described in any one of claims 1 to 12.
    [14]
    14. - The coated prosthesis according to claim 13, comprising:
    - a prosthesis with at least one surface of a biocompatible material selected from the group consisting of metallic, ceramic and polymeric;
    - a first coating comprising:
    or a sheet functionalized with reactive groups selected from the group consisting of amino (-NH2), carboxyl (-COOH), hydroxyl (-OH), sulfhydryl (-SH) and a combination of the above, deposited in at least a part of the surface of the prosthesis with an extension greater than 1cm2; Y
    or at least one organic molecule immobilized in the functionalized sheet; and - a second coating comprising pluri-potent or multi-potent cells adhered to the first coating.
    [15]
    15. - The prosthesis according to claim 14, wherein the immobilized organic molecule is fibronectin.
    [16]
    16. - The prosthesis according to any one of claims 13 to 15, wherein the cells adhered to the first coating are BM-MSC mesenchymal cells capable of differentiating into osteoblasts or chondrocytes.
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