ITVI20120210A1 - A PROCESS FOR THE IMPLEMENTATION OF A BIO-COMPATIBLE COATING FOR BONE PLANTS WITH BASIC MATERIAL EXTRACTED FROM BONE OF MAMMALS, AS WELL AS THE COVERING AND PLANT OBTAINED - Google Patents

Description

DESCRIPTION

Field of application

[001] The present invention is generally applicable to the technical field of materials for bone implants intended for osseointegration and specifically relates to a process for the production of a biocompatible coating for bone implants containing mammalian bones.

[002] The invention also relates to a coating and an implant obtained with the above process.

State of the art

[003] The prostheses and artificial grafts used for bone implants, for the anatomical and / or functional recovery of atrophic or traumatized parts, have been known from the earliest times. The first result to be obtained with this type of implant is the mechanical stability of the prosthesis or artificial graft, on which the success of the entire operation depends.

[004] Since the beginning of the 80s, Branemark's pioneering research has led to the discovery that some materials, among which the most used is titanium, when grafted into bone tissue give rise to a particular phenomenon , called “osseointegration”. This process is influenced by various physical parameters of the graft surface and is a biological process whereby a bone layer is intimately connected to the surface of the grafted object, endowing it with mechanical stability and solidity with respect to the surrounding bone.

[005] The characteristics of the graft surface capable of influencing the osseointegration process have been carefully studied in order to further improve its effectiveness to increase the aforementioned stability and, consequently, to make grafting possible stable of such objects even in conditions where the surrounding bone tissue is limited, not very dense or atrophic.

[006] This research has led to an in-depth study of two technological aspects: on the one hand, the production of surfaces with particular surface finishes, that is, with particular roughness and / or shape or size of the micro cavities created in the metal (and therefore, of the consequent degree of surface porosity), above all to facilitate cellular adhesion to metal, on the other hand the stimulation of cellular activity around the implanted object through the mechanical interaction of the surface as well as through the coating of said surfaces with materials other than the metal involved. These two types of surface modifications are often used simultaneously to maximize the final result.

[007] As regards the surface coating, both inorganic compounds (for example hydroxyapatite to mimic the composition of the mineral component of the bone tissue) and organic compounds (for example peptides, denatured collagen extracted from the dermis / tendon, to mimic the collagen component of bone tissue).

[008] From WO2011 / 154171 an endosseous implant is known, the external surface of which is at least partially covered with collagen, essentially with a structure of non-denatured native bone type I collagen. The document also describes a method of preparation of an intraosseous implant in which deposition is obtained with collagen in powder and in the presence of water, followed by dehydration of the implant by lyophilization. Deposition is carried out either by depositing an aqueous suspension of collagen powder on the implant or by depositing collagen dust on the wet implant.

[009] Although this implant has characteristics of high biocompatibility, it does not provide a veneering structure that guarantees the complete correspondence of the veneering material with the surrounding bone, lacking the covering of the mineral component characteristic of the bone itself combined with high biocompatibility. Furthermore, it does not yet possess all the specific biological properties favorable to regeneration at the graft site.

[0010] From US 2008/233203 a biomaterial is known comprising a biocompatible porous implant coated with a bone extract which penetrates into the pores of the implant. The biomaterial may also comprise a demineralized bone gelatin which can be deposited on the extract coat or mixed with the bone coat before being applied to the biocompatible implant. Although the bone biomaterial of this implant is characterized by remarkable stability, the bone extract of the veneering material is not fully biocompatible as it does not contain all the natural components of bone in the native state.

Presentation of the invention

[0011] The purpose of the present invention is to overcome the aforementioned drawbacks, by developing a bone coating for endosseous implants which has characteristics of complete physical, chemical and biological similarity with respect to the surrounding bone as well as high biocompatibility .

[0012] A particular aim is to conceive a natural coating for bone grafts that possesses specific biological properties favorable to regeneration at the graft site.

[0013] These aims, as well as others which will appear more clearly in the following, are achieved by a process for the production of a biocompatible coating for bone implants in accordance with claim 1.

[0014] In particular, the process comprises the preparation of a base material extracted from mammalian bone, deantigenation and possible demineralization of said base material in such a way as to maintain both quantitatively and qualitatively the content of Type I collagen in native conformation.

[001 5] The preparation of said base material comprises at least the steps of:

a) washing of the extracted bone material;

b) cutting of the material extracted and cut into desired shapes;

c) delipidization of the washed and cut material;

d) deantigenization of the delipized material;

in which the aforesaid phases are carried out with reagents, solvents and enzymes of type, concentration, and with application times chosen so as to avoid denaturing effects towards the Type I collagen in native conformation.

[0016] The material obtained with the process allows to obtain mutually correlated characteristics of mammalian bone which are advantageous from a clinical point of view.

[0017] The presence of a coating that simultaneously includes both bone components, the mineral one and the native collagen component, gives the coating pro-regenerative biological properties that favor the favorable outcome of the finished graft.

[0018] The bone replacement material obtained with the process according to the invention starting from mammalian bone tissue of any species is perfectly biocompatible and reabsorbable with physiological methods and times, contains bone collagen preserved in its native conformation and has the same mechanical properties of the unprocessed fabric.

The process further discloses a biocompatible lining and endoosseous implant in accordance with claims 8 and 10.

[0020] Further advantageous embodiments of the invention are defined in accordance with the dependent claims.

[0021] Further characteristics and advantages of the invention will become more evident in the light of the detailed description of some preferred but not exclusive embodiments of the coating for bone grafts.

Detailed description of some preferred embodiments [0022] The process for making the coating begins with the preparation of a base material extracted from animal bone, preferably from mammal. In theory, therefore, the base material could also be extracted from human bone.

[0023] In a preferred embodiment of the invention, the base material is subjected to the following steps:

a) washing of the extracted bone material;

b) cutting of the extracted material;

c) delipidization of the washed and cut material;

d) deantigenization of the delipized material;

[0024] According to the invention, the deantigenation step intended to eliminate the antigenic charge, however performed, must not alter some fundamental characteristics of the original tissue. First of all, it must not modify the cell-tissue interaction: even after the treatment, the cells of the bone tissue (osteoblasts and osteoclasts) must be able to both adhere to the treated tissue and to exert their biological action in a way no different from what they are able to do on untreated fabric.

[0025] Furthermore, and no less important, the deantigenation process must not alter in any way either the quantity or the native conformation of the bone collagen contained within the tissue of origin. This is because bone collagen is attributed not only to the property of giving the bone some of the mechanical characteristics it possesses, but also multiple biological properties favorable to the processes of bone regeneration and therefore of osseointegration. These properties, as it is known, depend on the protein being in its native state of conformation (they would in fact be lost, partially or wholly, if the protein were partially or completely denatured).

[0026] Therefore, in summary, the deantigenation step d) must be carried out in such a way as to keep the content of Type I collagen in native conformation unaltered both quantitatively and qualitatively, as well as not altering the surface properties of the mineral component.

[0027] To this end, it will be necessary to use instead of normal chemical reagents, whether they are inorganic solvents (acids or bases), organic solvents (acetone, etc) or enzymes, only those which, by their nature and chemical activity, do not extract the nor does collagen alter its native conformation, ie those having non-denaturing effects on collagen and which have no alteration effects on the mineral component.

[0028] All the preparatory steps, such as washing and delipidization, must also take place in conditions which neither remove nor denature the collagen and without altering the three-dimensional structure of the mineral part. Consequently, all steps a) to d) must be carried out at physiological temperature, preferably below 60 ° C to avoid denaturing the Type I collagen in its native conformation.

[0029] Conveniently, the bone tissue rendered free of antigens, before being applied to the implant to be coated, can undergo a phase e) of at least partial or complete demineralization. The purpose of demineralization is twofold: i) to modulate the resorption time of the coating once the coated object is grafted into the patient's bone tissue (greater demineralization will correspond to a shorter resorption time) and ii) partially expose or completely the bone collagen, which in the bone tissue is covered by bone apatite, in order to facilitate the biological actions ascribed to it described above.

[0030] This demineralization is preferentially carried out by immersing the fabric in a solution of HCI at a known concentration and stoichiometrically calibrated to obtain the desired degree of demineralization, finally adjusting the pH to neutrality by adding a suitable buffering solution.

[0031] In a preferential embodiment, the bone tissue thus treated, deantigenated and possibly fully or partially demineralized, will be subjected to a mechanical grinding step to produce a powder with a particle size preferably between 0.1 mm and 0.2 mm .

Of course, the result will not change with a lower particle size, for example between 100pm and 1000pm

[0032] Subsequently, the powder will be dispersed in a solvent or physiological solution to obtain a suspension

[0033] The powder will be suspended, preferentially but not necessarily at saturation, in water for injectable preparations or, preferably, in physiological solution.

[0034] The suspension will then be deposited on the surface of the object to be coated, for example by applying it by brush, spray, immersion or any other application method that allows homogeneous covering of the object.

[0035] The adhesion of the coating to the surface of the object must take place in conditions that do not alter either the mineral structure of the bone component or the protein conformation of the bone collagen contained therein. Preferably, adhesion will be obtained by subjecting the coated object to one or more freeze drying cycles.

[0036] As regards the principle of action of this coating, the scientific background can be summarized in data concerning other coatings of implantable objects, which aim to mimic the bone, with respect to which the present coating is a significant advance.

[0037] Other technologies, in fact, make it possible to coat the implantable objects intended to osseointegrate with substances that mimic the bone composition. One of these is certainly hydroxyapatite which is, due to its chemical composition and crystalline structure, similar to bone apatite. However, unlike bone apatite, its interaction with the cellular components of the bone tissue and specifically the osteoclastic component is different from that of natural apatite. As a result, hydroxyapatite coatings are destined to slowly reabsorb (with non-physiological kinetics) or not to reabsorb at all.

[0038] This entails potential problems such as the persistence of â € œ foreignâ € particles, however biocompatible, at the bone-implant interface, which, on the other hand, should be in complete contact with the patient's vital bone tissue, and the possible creation of a stable bond between the patient's bone tissue and the hydroxyapatite particle, that is the osseointegration not with the surface of the object, which is the desired outcome, but with the particle attached to it, with consequent possibility following the Application of the static and dynamic loads resulting from implantation - of the detachment of the particle from the surface of the object and, therefore, of the potential weakening of the binding forces that keep the object anchored to the receiving tissue.

In addition, the hydroxyapatite coatings do not possess any collagen component, unlike the coating in question. The presence of collagen, and moreover bone collagen and in native conformation in the coating in question, capable of exerting the biological effects ascribed to it, represents another substantial advance compared to hydroxyapatite coatings.

[0040] As regards the biological effects of native type collagen, these are widely documented in the biological and medical literature and range from positively mediating cell adhesion (both osteoblastic and osteoclastic), to induction of production and secretion of different growth factors by both cell types, to the increase in the expression and activity of enzymes key to osteoblastic activity, to the modulation of the activity of the receptors for the growth factors themselves, and others.

[0041] Last but not least, bone collagen is also a physical substrate for the mineralization of the bone tissue itself, acting as a set of nucleation centers for the formation of bone apatite crystals.

[0042] A bone implant using the coating described above comprises a support structure with an external surface, at least part of which, intended to come into contact with the human body following localization in situ, is covered with the coating and following the realization process described above.

[0043] Some examples of bone devices made with the coating according to the invention are given below.

[0044] Example 1 (in vitro study)

A titanium device was coated on the surface with bone coating obtained as described above. When the device was placed in contact with cells extracted from human bone marrow, a dose-dependent increase in vitality was detected (compared to a negative control consisting of an identical device without coating), up to a value of 1 , 6 times (+ 60%).

Cell viability was determined with a standard test, the so-called MTT assay where the acronym indicates the compound 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide. It is a standard colorimetric assay for measuring the activity of enzymes that reduce TTM to formazan, giving the substance a blue / purplish color; this occurs mainly in the mitochondria.

[0045] Example 2 (in vitro study)

A titanium device was surface coated as described above. When the device was placed in contact with cells extracted from human bone marrow, an increase in the release of pro-regenerative factors (TGF-beta1, TGF-beta2) by the cells themselves was induced in the cells themselves (compared to a negative control consisting of an identical device without coating), by 1.35 times (+ 35%). The concentration of TGF-beta1 and TGF-beta2 factors was measured through the ELISA (Enzyme-linked ImmunoSorbent Assay) test using commercial kits [0046] Example 3 (in vitro study)

A titanium device was coated on the surface as described above. When the device was placed in contact with osteoblastic cells grown from donor human bone, an increase in the activity of the alkaline phosphatase enzyme (typical marker of the metabolic activity of this cell type), compared to a negative control consisting of an identical device without coating, equal to 1.7 times (+ 70%). The activity of the alkaline phosphatase enzyme was determined through another colorimetric test, providing the cells with the substrate PNP (p-nitrophenyl phosphate) which is degraded by the enzyme into p-nitrophenol, a colorimetrically detectable compound.

[0047] Example 4 (in vivo animal study)

A titanium device was coated on the surface as described above. Once implanted in the femur of a laboratory animal (rabbit), it induced an increase in the final bone-implant contact surface (indicated as BIC, acronym for Bone Implant Contact) equal to 1.94 times (+ 94%) compared to the same uncoated device implanted in the contralateral femur, which is almost double.

[0048] Example 5 (in vivo animal study)

A titanium device was coated on the surface as described above. Once implanted in the femur of a laboratory animal (rabbit), it resulted in an increase in the early bone-implant contact surface (referred to as early BIC, and measured by administering specific fluorochrome to the animal on the day of implantation, detectable in the methacrylate resin bone sections containing the device subsequently explanted) equal to 3.7 times (+ 370%) compared to the same uncoated device implanted in the contralateral femur. The boneimplant contact surface (BIC) was measured by performing analysis of implant sections in a 45-day and 90-day coring of implants (coated and uncoated) implanted in animals. The cored sample, including the implant and a layer of surrounding bone, was embedded in methacrylate resin in order to perform micrometric sections which are then analyzed under the microscope. The animals were injected at different times with fluorescent markers (calcein green, xylenol orange, doxycycline, ...) which allow to distinguish the early bone deposition of the intermediate one from the long-term one. The data showed that the early osseointegration of the coated devices, in animal tests, induced by the bone coating, allows to obtain a bone-implant contact surface already equal to 78.8% of the implant at an early stage. Entire contact surface that would be obtained at the final explant time with the uncoated device.

[0049] Example 6 (observational clinical study)

Following the placement of 500 titanium dental implants coated with bone coating in patients candidates for prosthetic rehabilitation on implants, in the presence of bone tissue of quality D4 (classification according to: Lekholm U., Zarb G. A .: Kieferanatomie. In: Branemark P. I., Zarb G. A., Albrektsson T. (editors) Gewebeintegrierter Zahnersatz - Osseointegration in klinischer Zahnheilkunde. Quintessenz, Berlin Chicago London, 1985, pp. 197-199), or D4 and D5 according to the Misch classification (Misch C.E., Density of bone: effect on treatment plans, surgical approach, healing, and progressive bone loading, Int. J. Orai Implantol. 1990, 6 (2), 23-31) A 4-6 month success rate of 97.7% was found (in the most conservative hypothesis, attributing all failures to the sole failure of osseointegration of the implant). Analyzing the failed cases, possibly attributable to different causes (surgical error, infection, non-responsive patient), the success rate could be considered equal to 99.1%.

The parameters of success (or failure) to implant consist of the percussion test (qualitative test), the unscrewing test (counter-torque test) and the radiographic evaluation as described by Albrektsson and Zarb (Albrektsson T, Zarb G., Worthington P. , Erikkson A.R., â € œThe long-term efficacy of currently used dental implants: a review and proposed criteria of successâ €, Int. J. Orai Maxillofac. Implants 1986, 1, 11-25. The implant must have mechanical stability and the amount of pre-implant bone resorption must not be greater than 1.5 mm in the first year and 0.2 mm in the following years.

[0050] Example 7 (clinical study)

25 implants with a sandblasted and acidified surface were coated with said bone coating and positioned immediately after dental extraction in sites corresponding to molar teeth or second pre-molars, where the placement of immediate post-extraction implants is one of the associated conditions at greater risk of implantation failure. Following placement, no adverse effects caused by the applied bone coating were observed. Follow-up clinical and radiographic controls performed at 4, 6 and 12 months later showed the acquisition of osseointegration by all implants. At 12 months, implantation success was 95.8% and implant survival was 100%.

[0051] Studies clearly show that the invention has achieved the aim of creating an improved endoosseous implant that allows for better osseointegration of the same.

Claims (10)

CLAIMS 1. A process for the production of a biocompatible coating for bone implants, comprising the preparation of a base material extracted from mammalian bone, deantigenation and possible demineralization of said base material in such a way as to keep the collagen content unaltered both quantitatively and qualitatively Type I in native conformation. 2. Process according to claim 1, wherein the preparation of said base material comprises at least the steps of: e) washing of the extracted bone material; f) cutting of the extracted material; g) delipidization of the washed and cut material; h) deantigenization of the delipized material; in which said phases are carried out with reagents, solvents and enzymes of type, concentration, and with application times chosen so as to avoid denaturing effects towards Type I collagen in native conformation. 3. Process according to claim 2, in which said steps a), b) and c) are carried out at physiological temperatures below about 60 ° C so as to preserve the native structure of Type I collagen unaltered. 4. Process according to claim 3, in which said base material is subjected at least partially to a step e) of demineralization by means of chemical reagents with concentration, treatment times and temperatures such as to preserve the native structure of Type I collagen unaltered. 5. Process according to claim 4, wherein said extracted, deantigenated, optionally demineralized base material is ground to obtain a powder with a particle size between 0.1 mm and 0.2 mm. 6. Process according to claim 5, wherein said powder of base material is dispersed in a suitable liquid solvent so as to obtain a suspension applicable to at least part of the external surface of a bone implant. 7. Process according to claim 6, wherein said liquid suspension applied to a bone implant is subjected to dehydration by lyophilization to form the finished coating. 8. A biocompatible coating for bone implants, comprising a natural base material extracted from processed mammalian bone according to one or more of the preceding claims, in which powdered ground base material is the solid part of a brush applicable suspension, to spray, by immersion or any other method to at least part of the external surface of a bone implant, in which said base material is suitably deantigenated, maintaining both quantitatively and qualitatively the content of Type I collagen in native conformation with respect to that present in the ™ Initial Mammalian Bone Extract. The biocompatible coating according to claim 8, wherein the liquid suspension applied to at least part of the outer surface of a bone implant is lyophilized to form the finished coating. 10. A biocompatible bone implant, comprising a support structure with an external surface, in which at least part of said external surface intended to come into contact with the human body as a result of localization in site is covered with a coating according to one of the claims 8 and 9, using a manufacturing process according to one or more of claims 1 to 7.