COVERING AND/OR IMPREGNATING METHOD

TECHNICAL FIELD

The invention refers to a covering and/or impregnating method suitable for covering surfaces of a shaped body.

In particular, these shaped bodies comprise medical or biomedical products, including materials suitable for making implants and prostheses.

In the field of implant materials, the use is known of techniques suitable for covering said bodies to make them biocompatible or bioactive.

By "biocompatible" is meant the capacity of a foreign body to be accepted by an organism with which it comes into contact.

A good biocompatible material does not cause negative uncontrollable reactions and can therefore be surgically implanted with limited or no contraindications or undesired effects.

An implant material must however possess further characteristics, including mechanical properties suitable for the function it has to perform once implanted. In the field of orthopaedic prostheses materials are already used such as metals, alloys, ceramics, polymers and bioglasses.

To each material correspond specific characteristics, the evaluation of which leads to the choice of the product most suitable for each specific use. If necessary these materials can be combined.

TECHNOLOGICAL BACKGROUND.

Processes are known for example suitable for covering alloys or metals with a covering suitable for upgrading their biocompatibility and their bioactivity; this covering is, for example, of the ceramic type.

In this case, the ceramic can be made up of hydroxyapatite or by-products, already used because they are very similar to the biological structure of the bone. Hydroxyapatite is a mineral belonging to the apatite family containing calcium phosphates and represents one of the fundamental components of the bone. Hydroxyapatite thus provides a good bio substrate for the adhesion and the proliferation of the bone cells, allowing the organism to incorporate the prosthesis and promote the formation of new bone tissue.

The metal on the other hand provides a support with high mechanical properties.

For example, in a procedure for obtaining the deposition of the ceramic layer, the ceramic undergoes high temperature to make it liquid and then apply it to the prosthesis.

These high temperatures can be obtained using plasma torches that heat a gas up to

2O ₅ OOO ⁰ C.

Once liquefied, the ceramic is sprayed on the prostheses, covering this, and is then subsequently cooled.

This state of the art, however, does have some drawbacks.

A first drawback is that the liquefying of the ceramic produces great energy expenditure and the use of extremely costly machinery.

A second drawback is that the spray application does not allow uniform spreading of the ceramic: in fact, the deposited layer has variations in thickness on different points of the support.

Another drawback is that the application of the covering is made particularly difficult by the presence of irregularities or nanocavities: in these areas, the spray does not manage to reach all the points of the prosthesis surface with the consequent risk of a non-uniform or incomplete covering.

OBJECTS OF THE INVENTION. An object of the invention is to upgrade the prior art.

Another object of the invention is to develop a process suitable for producing a covering of protein material, that can be reliably, easily and quickly applied and which is cost effective, and has the capacity to favour the adhesion and the proliferation not only of the bone cells, but also of those of other cellular phenotypes, stimulating the formation of new tissue and giving the covered material characteristics of biocompatibility and anti-thrombogenicity.

According to one aspect of the invention, a covering method is provided suitable for covering surfaces of a shaped body, comprising: placing an electrolytic solution containing protein material in an electrochemical cell; producing a difference in potential between electrodes of said electrochemical cell generating a flow of protein material towards at least one of said electrodes; placing said shaped body in said

electrochemical cell in such a way that said flow touches said surfaces, covering them with said protein material, characterized in that said protein material comprises fibroin.

According to another aspect of the invention, a shaped body is provided that comprises a covering of protein material, characterized in that said protein material comprises fibroin.

Consequently, the covering method suitable for covering surfaces of a shaped body permits depositing a protein material on the shaped body, giving it innovative technological characteristics and numerous advantages in terms of use. A first advantage of the invention is that the covering material is made up of biological material, and therefore similar to the structures of the organism with which it comes into contact.

A second advantage of the invention is that the fibroin, in particular, is known for its biocompatibility characteristics, i.e. it is well tolerated by the organism. A third advantage is supported by various studies that have shown that the amino acid sequences of fibroin favour cell adhesion and proliferation, prompting the regeneration of the tissues close to the implant site and cellular colonisation. A further advantage of the invention is that the method can be easily applied on metal- based prostheses or implant materials or which in any case comprise conducting materials or, again, also on non-conducting porous materials.

Another advantage of the invention is that the covering, once the protein material has deposited, can be stabilised to upgrade its mechanical characteristics. A further advantage of the invention is that the covering of protein material can be made variably porous according to specific requests to upgrade its predisposition to cellular colonisation, and thus favour specific processes.

An additional advantage of the invention is that uniform coverings of protein material are also obtained on irregular surfaces; in particular, porous shaped bodies can be obtained impregnated with fibroin on which the inner surface cavities are also covered with protein material.

Another advantage is that conductor bodies of any shape and size can be covered. A further advantage of the invention is that the entire process is performed with low

energy expenditure and without using costly machinery.

Another advantage of the invention is that the covering of protein material can be associated with additives of various kinds, including biologically active molecules such as cellular growth and adhesion factors, or drugs.

Another advantage is that depositing protein material on a rigid surface allows to overcome the problems related to the sensitivity of the cells to local rigidity, creating a softer layer on which the cells can proliferate and in which nutrients and metabolic substances can spread.

A further advantage of the invention is that the covering of protein material can comprise other proteins besides fibroin or other natural or synthetic molecules with electric charge in non-null solution.

EMBODIMENTS OF THE INVENTION.

Further characteristics and advantages of the present invention will appear more evident from the detailed description of a preferred but not exclusive embodiment of a covering method suitable for covering surfaces of a shaped body, illustrated below indicatively by way of non limiting example.

The method according to the invention entails placing an electrolytic solution containing protein material, dissolved or in suspension, in an electrochemical cell. This protein material has electrophoretic properties, meaning it has the capacity to move in the electrolytic solution towards an electrode with an appropriate charge. The electrochemical cell comprises a generator able to produce a difference of potential between the electrodes.

The electrodes of the electrochemical cell are made up of electric conductor bodies made, for example, with metals, graphite, conducting oxides, conducting polymers and any conducting material, and suitable for being immersed in the electrolytic solution.

After having produced an adequate electric field, the protein material starts to migrate towards the electrode with appropriate charge, generating a flow of protein material towards this; this flow is therefore the upshot of the movement of all the charged molecules in the electrolytic solution towards the opposite electrode. According to the invention, the placing is further provided of a shaped body immersed

in the electrolytic solution and placed between the electrodes or in any case in such a way that the flow of protein material laps or cuts into or touches its surfaces, covering these with the protein material.

On the surfaces of the conductor body, in fact, weak type chemical interactions are established between the surfaces and protein, and also between protein and protein, permitting the formation of a layered covering of protein material.

The weak chemical interactions that are established are, for example: Van der Waals forces, hydrogen bonds, dipole moments, hydrophobic or electrostatic interactions.

The shaped body is a product substantially, but not only, intended for medical or biomedical use, such as for example supports for cell cultivation, implant materials and prostheses.

When the shaped body on which to make the deposition is made up of an electric conducting material, it can itself represent one of the two electrodes, allowing the direct deposit of the fibroin on the surface of the shaped body; in this case therefore, it is enough to place the shaped body in the solution and this will act as an electrode and its counter-electrode, both connected to the respective poles of the battery.

This method thus permits covering metal prostheses or prostheses made up of any conducting material by simply replacing the electrode on which deposition occurs with the prosthesis.

In the particular embodiment in which the shaped body is not an electric conductor, the deposition of fibroin is obtained by placing at least one portion of the shaped body in a substantially transversal position with respect to the direction of migration of the protein material; in migrating, the molecules in solution encounter the surface of the shaped body and deposit on this.

Alternatively, one of the two electrodes can be fitted, completely or partially, inside the shaped body, before being removed or also left inside the shaped body at the end of the covering process.

In the particular case in which the shaped body comprises a porous matrix, it occurs that the flow of protein material can also migrate inside the pores and the cavities thus impregnating the shaped body; in fact, the fibroin also covers the surface of the inner interstices, obtaining a porous material impregnated with fibroin with excellent

biological properties.

In this case as well, the matrix, if made of electric conducting material, can itself be the electrode, otherwise it can be placed between the two electrodes.

According to the invention, the electrolytic solution is made up of water in which is dissolved a quantity of fibroin between 0.1% and 15%; it has been ascertained that the best results are obtained with concentrations of fibroin of around 1%.

To the electrolytic solution can also be added proteins different to fibroin or other different molecules to obtain a covering of mixed protein material.

The power generator maintains a difference in potential between the electrodes between 0 and 40 V, nevertheless this interval can be extended according to need.

Increase in voltage is matched by an increase in the deposition speed but also in the production of gas bubbles or other undesired effects, for example, the formation of a non-uniform covering, deriving from the presence of these bubbles or from alterations of the components used in the process; for this reason, the best difference in potential is between 1 V and 5 V.

The pH of the solution determines the direction of the migration of the protein material in solution; in particular, when the process occurs in a pH value interval between 6 and 7, the fibroin migrates towards the positive electrode.

The electro-deposition process occurs at room temperature.

The distance between the two electrodes can vary from just a few millimetres to several centimetres, bearing in mind that the shorter the distance, the higher the deposition speed.

To upgrade process efficiency, the number and the shape of the electrodes that can be immersed in the solution can vary.

The start of the power generator produces an electric current deriving from the passing of the electrons exchanged with the electrodes in the processes and reductions of the molecules and/or of the ions present in solution, as is supported by the continuous flow of these molecules and/or ions towards the electrodes.

The quantity of deposited protein material therefore depends on different variables such as: applied voltage, concentration and volume (for very small volumes, an impoverishment of the reagents can occur) of the initial solution, pH, electrode

material and their distance.

Coverings of different thickness can be obtained, that can reach hundreds of microns. According to the invention, the covering of the protein material obtained after deposition on the shaped body can be stabilised to make it stable and insoluble in water, and if necessary dry it by means of drying or freeze-drying. It is known that, in its soluble form, fibroin is found in the random coil and a-helical structures, which can also remain at the time of deposition on the shaped conductor body.

To make the covering of protein material insoluble after deposition, a modification is induced of the fibroin structure towards a more stable and insoluble conformation. For example, to stabilise the covering of protein material obtained by deposition, the transformation can be induced of the fibroin towards the β sheet shape by treating this covering with an organic solvent, i.e. by immersing the surface of a conductor body covered with fibroin in a solution containing water and at least one organic solvent in a percentage between 20% and 100% (normally between 60% and 80%) for a predetermined time interval between 1 and 24 hours. This organic solvent is for example a hydrophilic alcohol such as methanol. By increasing the concentration of solvent and/or the treatment times a greater variation is induced of the fibroin structure towards the β sheet crystal shape with consequent greater stabilisation.

Alternatively, the covering of protein material can be stabilised by treating it with water vapour, meaning placing the covered shaped bodies in an environment saturated with water vapour for a predetermined time interval such as to induce the crystallisation of the fibroin, and which is normally between 1 and 24 hours. After depositing the fibroin, the covering of protein material can undergo a freeze- drying process.

In this case, the deposited protein material undergoes a transformation from a more or less compact gel to a sponge with controlled porosity.

This covering sponge shape, besides being preferable to the gel one for certain uses, can increase practicality and make its preservation easier in conditions of sterility. To obtain this drying, the freeze-drying technique is used; the covering temperature of

protein material is lowered to -2O ⁰ C or -80 ⁰ C freezing the liquid component, and then the ice thus obtained is sublimated under vacuum.

After the deposition, in particular as the last working phase, the covering of protein material can be sterilised using any of the chemical or physical means known to the technician in the field such as, for example γ rays.

Fibroin is a protein obtained by separation and purification from the cocoon of the larva of a number of organisms, such as, for example Bombyx mori.

Alternatively, fibroin can be used taken from various breeds of Bombyx mori, from other lepidopters, from anthropods, from genetically modified organisms and in general from other biological or synthetic sources.

The processes and the products obtained using fibroin with different molecular weight or amino acid sequence different from the fibroin obtained from Bombyx mori can therefore be considered equivalent.

The presence of fibroin in a support for the cellular growth or implant, whether this is used for cell growth in vitro or in vivo or for the regeneration of tissues, improves the support's capacity to ensure the proliferation of the cells themselves without causing an immune reaction or extending the inflammatory reaction; this effect is thought to be determined by amino acid sequences favourable to the adhesion and the activation of the cellular metabolism.

The covering of protein material can be associated, for purposes of improving the biological characteristics, with additives comprising biologically active molecules, including cellular growth or adhesion factors, or antibiotics, anti-inflammatories, etc...