WO2014024171A1

WO2014024171A1 - Process for preparation of a biocompatible coating for bone grafts as well as coating and implant obtained therewith

Info

Publication number: WO2014024171A1
Application number: PCT/IB2013/056527
Authority: WO
Inventors: Stefano PAGNUTTI; Antonio Gigante
Original assignee: Akimedix S.R.L.
Priority date: 2012-08-10
Filing date: 2013-08-09
Publication date: 2014-02-13
Also published as: EP2882463A1; ITVI20120210A1

Abstract

A process for preparation of a biocompatible coating for bone implants comprises the steps ofextracting a base material from mammal bone, de- antigenating and possibly demineralizing the base material,to maintain the Type I native collagen content unaltered both in quantity and in quality, mechanically grinding the base material to obtain a powder and dispersing the powder in a liquid solvent selected from the group comprising water and salines to obtain a suspension adapted to be directly applied to a bone implant. A biocompatible coating for bone implants comprises a base material extracted from mammal bone which has undergone a process in which the base material grinded into powder is the solid component of a suspension that is applied by brush-painting or dipping to at least part of the surface of a bone implant, wherein said base material is conveniently de-antigenated to maintain the native Type I collagen content unaltered both in quantity and in quality with respect to that in the initial mammal bone extract.

Description

PROCESS FOR PREPARATION OF A BIOCOMPATIBLE COATING FOR BONE GRAFTS AS WELL AS COATING AND IMPLANT OBTAINED THEREWITH

DESCRIPTION

Field of the invention

[001 ] The present invention generally finds application in the technical field of materials for bone implants designed for osseointegration and namely relates to a process for realizing a biocompatible coating for bone implants containing mammal bones.

[002] The invention further relates to a coating and an implantobtained with said process.

Background art

[003] Prosthesis and artificial grafts used for bone implants, for anatomical and/or functional recovery of atrophic and traumatized parts, have been known for a very long time. The first desired goal of this kind of implants is the mechanical stability of the prosthesis or the artificial graft, ensuringsurgical success.

[004] Since the beginning of the eighties, the pioneering studies of Branemark led him to discover that some materials, among which the titanium is the most used, when grafted in the bone tissue originate a particular phenomenon called "osteointegration". This process is affected by various physical parameters of the implant surface and is a biological process whereby a bone layer is intimately joined to the surface of the grafted object, and imparts thereto mechanical stability and solidity with respect to the surrounding bone.

[005] The characteristics of the graft surface that can affect the osseointegration process have been carefully studied to further improve its effectiveness by enhancing such stability and, as a result, to afford stable grafting of such objects even where the surrounding bone tissue has a limited extension, a poor density or is , atrophic.

[006] This study has led to analyze two technological aspects: on one hand the production of surfaces having a particular surface finish, namely a particular roughness and/or shape or size of the micro cavities formed in metal, (and accordingly a particular degree of surface porosity), to especially facilitate cellr adhesion to metal, on the other hand the stimulation of cell activity around the grafted object through mechanical surface interaction as well as by coating said surfaces with materials other from the relevant metal. These two types of surface modifications are often simultaneously used to optimize the final result.

[007] Concerning surface coating, both inorganic compounds (e.g. hydroxyapatite to mimic the composition of the mineral part of the bone tissue) and organic compounds (e.g. peptides, denatured collagen extracted from dermis/tendon to mimic the collagen part of the bone tissue) have been used.

[008] From WO201 1 /1541 71 an endosteal implant is known, whose outer surface is covered at least partially with collagen, essentially having a structure of native non denatured Type I bone collagen. The document further describes a method for preparing an endosteal implant in which deposition is obtained with powdered collagen and in the presence of water, followed by dehydration of the implant through lyophilization. Deposition is obtained either by deposition of an aqueous suspension of collagen powder onto the implant or by deposition of collagen powder onto the wet implant.

[009] While this implant shows high biocompatibility, it doesn't provide a coating structure that guarantees full matching of the coating material and the surrounding bone, due to the lack of a coating of the mineral part, which isthe characteristic of the bone itself, in addition to high biocompatibility. Furthermore, it still doesn't have all the special biological properties that promote regeneration in the graft site.

[010] US2008/233203 discloses a biomaterial comprising a biocompatible porous graft coated by a bone extract which penetrates the pores of the graft. The biomaterial may also comprise a demineralized bone gelatin which can be laid down onto the extract coating or mixed with the bone coating before being applied to the biocompatible graft. Although the bone biomaterial of this graft is characterized by remarkable stability, the bone extract of the material of the coating is not fully biocompatible because it doesn't contain all the natural components of the bone in the native state.

Disclosure of the invention

[001 1 ] A general object of the present invention is to eliminate or at least reduce the above drawbacks, by providing a bone coating for endosteal grafts that exhibits complete biological, chemical and physical similarity with the surrounding bone as well as high biocompatibility.

[0012] A particular object is to provide a natural coating for bone grafts that has specific biological properties favorable for regeneration in the graft site.

[0013] These and other objects, as better explained hereinafter, are fulfilled by a process for preparing a biocompatible coating for bone implants as defined in claim 1 .

[0014] In particular, the process comprises the steps ofpreparing a base material extracted from mammal bone, optionally demineralizing it to maintain the native Type I collagen content unaltered both in quantity and in quality, mechanically grinding the base material to obtain a powder and dispersing the powder in a liquid solvent.

[0015] The process is characterized in that the powder is dispersed in a liquid solvent selected from the group comprising water and salines, to obtain a suspension that is applied directly to at least a part of the outer surface of a bone implant with no filtration or other intermediate treatment step.

[0016] The step or preparing the base material comprises at least the steps of:

a) washing the extracted bone material;

b) cutting the extracted material to desired shapes;

c) delipidating the washed and cut material;

d) de-antigenating the delipidated material;

wherein said steps are carried out at physiological temperatures of less than about 60 °C and with reagents, solvents and enzymes whose type, concentration and application times are selected in view of avoiding denaturing effects toward the native Type I collagen.

[0017] The material obtained from this process provides mutually correlated clinically advantageous properties of the mammal bone.

[0018] The presence of a coating simultaneously comprising both bone components, the mineral one and the collagen one having a native conformation provides the coating with pro-regenerative biological properties, promoting the success of the finished graft.

[0019] The replacement material of the bone as obtained with the process of the invention from a mammal bone tissue of any type proves to be perfectly biocompatible and absorbable in physiological manners and at physiologicaltimes, contains bone collagen in its native conformation and has the same mechanical properties as the unprocessed tissue.

[0020] The invention also provides a coating and a biocompatible endosteal implant as defined in claims 10 and 1 1 .

[0021 ] Further advantageous embodiments of the invention are defined in accordance with the dependent claims.

[0022] Further characteristics and advantages of the invention will be more apparent from the detailed description of a few preferred, nonexclusive embodiments of the coating for bone grafts.

Detailed description of a few preferred embodiments

[0023] The process for preparing the coating starts with the preparation of a base material extracted from animal bone, preferably mammal bone. So, theoretically, the base material could be extracted from human bone too.

[0024] In a preferred embodiment of the invention, the base material undergoes to the following steps:

a) washing the extracted bone material;

b) cutting the extracted material;

c) delipidating the washed and cut material;

d) de-antigenating the delipidated material.

[0025] According to the invention, the de-antigenating step, which is designedto eliminate the antigenic load, however it is carried out, must not change some of the basic properties of the original tissue. First it must not alter the cell-tissue interaction: even after treatment, the cells of the bone tissue (osteoblasts and osteoclasts) should be able to both adhere to the treated tissue and exert their biological action in the same way as they do on the untreated tissue.

[0026] Also, and not the least, the de-antigenation process must not alter in any manner either the quantity or the native conformation of the bone collagen contained in the original tissue. This is because the collagen has multiple biological properties that are favorable for bone regeneration processes and hence for osseointegration, beyond those of imparting to the bone some of the its inherent mechanical properties. Such properties, as is known, are dependent on the fact that the protein is in its native conformation (they would be partially or totally lost, if the protein were partially or entirely denatured).

[0027] Therefore, in brief, the de-antigenation step d) shall be carried out to maintain the native Type I collagen content unaltered both in quantity and in quality, as well as to avoid alteration of the surface properties of the mineral component.

[0028] For this purpose, instead of the usual chemical reagents, i.e. inorganic solvents (acids or bases), organic solvents (acetone, etc.) or enzymes, the only chemical reagents that may be used are those that do notextract collagen by their nature and chemical activity, and especially do not change its native conformation, namely those having non-denaturing effects toward collagen and no altering effect on the mineral component.

[0029] Also all the preparatory phases, such as washing and delipidation shall be carried out in conditions that do not remove nor denature collagen and without altering the three-dimensional structure of the mineral component. As a result, all the steps from a) to d) shall be carried out at physiological temperature, preferably below 60 °C, to avoid denaturation of the native Type I collagen.

[0030] Conveniently, the antigen-free bone tissue so obtained, before being applied to the implant to be coated may undergo a step e) of at least partial or full demineralization. Demineralization has a dual purpose: i) modulate the absorption time of the coating when the coated object is grafted in the patient bone tissue (a shorter absorption time will correspond to a higher demineralization) and ii) partially or completely expose bone collagen, which is coated by bone apatite in bone tissue, to facilitate its above described biological actions .

[0031 ] Such demineralization preferably obtained by dipping the tissue in a HCI solution with a known and stoichiometrically calibrated concentration to obtain the desired demineralization degree, and eventually adjusting the pH to neutrality through the addition of a suitable buffer solution.

[0032] In a preferred embodiment the bone tissue so treated, de- antigenated and possibly partially or entirely demineralized, will undergo a grinding phase to obtain a powder with a particle size preferably ranging from 0.1 mm to 0.2 mm. Of course, the result will not change with a lower particle size, e.g. ranging from 1 00 μιη to 1 000 μιη.

[0033] Afterwards, the powder will be dispersed in a solvent or in a physiological solution for obtaining a suspension.

[0034] The powder will be suspended, preferably but not necessarily to saturation, in water for injectable preparations or, preferably, in a saline.

[0035] The suspension will be directly applied to at least a part of the outer surface of the bone implant to be coated, without any intermediate filtration or treatment step.

[0036] The suspension may be applied, for example, with a method selected from the group comprising brush or spray painting, dipping or any other application mode allowing the object to be homogeneously coated.

[0037] The adhesion of the coating to the surface of the object must occur in conditions that change neither the mineral structure of the bone component nor the protein conformation of the bone collagen contained therein. Preferably, adhesion will be obtained by submitting the coated object to one or more lyophilization cycles.

[0038] Concerning the principle of action of such coating, the scientific background can be summarized to data relating to other coatings of implantable objects, which have the purpose to mimic bone, according to which the present coating is a remarkable development.

[0039] Other technologies, allow the implantable objects designed for osseointegration with substances that mimic the bone composition. One of these is certainly hydroxyapatite which is similar to bone apatite, in terms of chemical composition and crystalline structure, . Nevertheless, unlike bone apatite, the interaction with the cell component of the bone tissue, namelythe osteoclastic component is different from that of natural apatite. Therefore, hydroxyapatite coatings are likely to be reabsorbed slowly (with non- physiological kinetics) or not to be reabsorbed at all.

[0040] This may cause potential problems, such as the permanence of'foreign" particles, although biocompatible, at the bone-implant interface, which interface should be entirely contact the vital bone tissue of the patient , and the possible creation of a stable connection between the bone tissue of the patient and the hydroxyapatite particle, which means that the osseointegration doesn't occur with the object surface, as it would be desired, but with the particle adhered thereto and this may cause, upon application of static and dynamic loads after implamtation, the detachment of the particle from the object surface and so, the potential weakening of the bonding strengths which maintain the object anchored to the receiving tissue.

[0041 ] Moreover, unlike the coating in the present document, the hydroxyapatite coatings have no collagen component, . The presence of collagen, and namely the presence of native bone collagen , with its biological effects , is another substantial development with respect to hydroxyapatite coatings.

[0042] As for the biological effects of native Type I collagen they are well documented in the biological and medical literature and they include positively mediating cell adhesion (of both osteoblasts and osteoclasts), inducing production and secretion of various growth factors by both cell types, increasing expression and activity of key enzymes in osteoclastic activity, modulating the activity of receptors for the growth factors, and more. [0043] Not the least, collagen is also a physical substrate for mineralization of the bone tissue, and acts as a group of nucleation centers to form bone apatite crystals.

[0044] A bone implant that uses the above described coating comprises a support structure with an outer surface, at least the part of the latterdesigned to contact the human body after on-site location, being covered with the coating according to the above preparation process.

[0045] A few examples of bone devices formed with the coating of the invention will be described below.

[0046] Example 1 (in vitro study)

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

Cell vitality was determined by a standard test, the so-called MTT assay, which stands for the compound 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide. This isa colorimetric assay that measures the activity of the enzymes which reduce MTT to formazan while imparting a blue/purple color to the substance; this mostly happens in mitochondria.

[0047] Example 2 (in vitro study)

The surface of a titanium device was coated as described above. When the device was put in contact with cells extracted from human bone marrow, the release of pro-regenerating factors (TGF-beta1 , TGF-beta2) in cells was increased by the cell themselves (as compared with a negative control consisting of an identical device without coating) by 1 .35 times (+35%). Concentration of the TGF-beta1 e TGF-beta2 factors was measured by ELISA (Enzyme-linked Immuno Sorbed Assay) using commercial kits.

[0048] Example 3 (in vitro study)

A titanium device was coated as described above. When the device was put in contact with osteoblast cells cultured from sampling of human bone of a donor, a 1 .7 time-increase (+70%) increase in the activity of alkaline phosphatase (a typical marker of metabolic activity of this cellular type) was observed in cells, as compared with a negative control consisting of an identical device without coating. The activity of the alkaline phosphatase enzyme was determined through another colorimetric test, which consisted in supplying cells with the substrate PNP (p-nitrophenilphosphate), which is degraded by the enzyme to p-nitrophenol, a colorimetrically observable compound.

[0049] Example 4 (in vivo animal study)

The surface of a titanium device was coated as described above. Once it was implanted in the femur of a laboratory animal (rabbit), it caused a 1 .94-time increase (+94%), ofthe final bone-implant contact surface (BIC, Bone Graft Contact), i.e almost twice, as compared with an identical uncoated device implanted in the contralateral femur.

[0050] Example 5 (in vivo animal study)

The surface of a titanium device was coated as described above. Once it was implanted in the femur of a laboratory animal (rabbit),, it caused a 3.7-time increase (+370%) of the early bone-implant contact surface (referenced to as early BIC, and measured by administering a specific fluorophore to the animal on the implantation day, as observed in bone sections made of methacrylate resin containing the device later explanted) as compared with an identical uncoated device implanted in thecontralateral femur. The bone- implant contact surface (BIC) was measured by analyzing sections of implants (coated and uncoated) in animals with probes taken at days 45 and 90. The probed sample, comprising the implant and the surrounding bone layer, has been included in methacrylate resin to obtain micrometric sections later analyzed by microscope. Fluorescent markers (calcein green, xylenol orange, doxycycline), which highlight early bone deposition as compared with long-termdeposition, were injected to animals at different times. Data shows that early osseointegration of coated devices induced by the bone coating, in tests on animals, already provide at early times, a contact bone-implant surface that is 78.8% the entire contact surface which would be obtained at the final stage of the explant with an uncoated device. [0051 ] Example 6 (observational clinic study)

Upon implantation of 500 titanium dental implants coated with the bone coating in patients candidate for prosthetic rehabilitation on implants, in the presence of D4 quality bone tissue (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, 1 985, pp. 1 97-1 99), or D4 and D5 quality tissue according to Misch classification (Misch C.E., Density of bone: effect on treatment plans, surgical approach, healing, and progressive bone loading, Int. J. Oral Graftol. 1 990, 6(2), 23-31 ) a success percentage of 97.75% has been observed at 4-6 months (in the most conservative hypothesis, attributing all the failures only to failed osseointegration of the implant). Upon analysis of failure cases, which may be attributed to different causes (surgical error, infection, non-responsive patient) the success percentage could be 99,1 %.

The parameters for implant success (or failure) consist in a percussion proof (quality test), an unscrewing proof (reverse torque test) and in X-ray assessment as described in Albrektsson and Zarb (Albrektsson T., Zarb G., Worthington P., Erikkson A. R., "The long-term efficacy of currently used dental implants: a review and proposed criteria of success", Int. J. Oral Maxillofac. Implants 1986, 1 , 1 1 -25. The implant must exhibit mechanical stability and the pre-implant bone re-absorbance entity mustn't be higher than 1 .5 mm in the first year and 0.2 mm in the following years.

[0052] Example 7 (clinical study)

25 grafts a sandblasted and acidified surface have been coated with said bone coating and placed immediately after dental extraction in sites corresponding to molar or second pre-molar teeth, i.e. where positioning of immediate post-extraction implants is one of the conditions associated with a higher failure risk. After positioning, no negative effect caused by the applied bone coating was observed. Clinical and X-ray follow-ups made after 4, 6 and 1 2 months showed osseointegration of all the implants. At 1 2 months implantsuccess scored 95,8% and implant survival was 1 00%. [0053] These studies show with evidence that the invention has reached the goal of creating a perfected endosteal graft, allowingimproved osseointegration thereof.

Claims

1 . A process for preparation of a biocompatible coating for bone implants, which comprises the steps of preparing a base material extracted from mammal bone, optionally demineralizing said base material to maintain the native Type I collagen content unaltered both in quantity and quality, mechanically grinding said base material to obtain a powder and dispersing the powder in a liquid solvent,

characterized in that said powder of base material is dispersed in a liquid solvent selected from the group comprising water and salines to obtain a suspension, said suspension being directly applied to at least part of the outer surface of a bone implant without intermediate filtration or treatment step.

2. A process as claimed in claim 1 , wherein the particle size of said powder ranges from 0.1 mm to 0.2 mm.

3. A process as claimed in claim 1 , wherein the application of said suspension is carried out with a method selected from the group comprising the laying down by brush or spray painting or dipping.

4. A process as claimed in claim 1 , wherein the preparation of the base material comprises at least the steps of:

a) washing the extracted bone material;

b) cutting the extracted material;

c) delipidatiing the washed and cut material;

d) de-antigenating the delipidated material.

5. A process as claimed in claim 4, wherein said steps from a) to d) are carried out at physiological temperatures of less than about 60 °C and with reagents, solvents and enzymes whose type, concentration and application times are adapted to avoid denaturing effects toward native Type I collagen.

6. A process as claimed in claim 4, wherein said base at least partially undergoes a demineralization step e) of by dipping of said base material in a

HCI solution.

7. A process as claimed in claim 6, wherein said HCI solution has a known and stoichiometrically calibrated initial concentration.

8. A pocess as claimed in claim 7, wherein a suitable buffer solution is added to said HCI solution, to adjust thepH to neutrality.

9. A process as claimed in claim 1 wherein, once said liquid suspension has been applied nto a bone implant, it undergoes dehydration by lyophilization to form the finished coating.

1 0. A biocompatible coating for bone implants, comprising a layer of a base material deposited on at least part of a bone implant, said base material being obtained through a process as claimed in one or more of the preceding claims and having a native Type I collagen content unaltered with respect to that in the initial mammal bone extract, wherein said layer of base material is lyophilized to form the finished coating.

1 1 . A biocompatible bone implant, comprising a support structure with an outer surface, wherein at least the part of said outer surface that is designed to contact the human body upon on-site implantation is coated with a coating as claimed in claim 1 0 using a process for preparation of the base material as claimed in one or more of claims from 1 to 9.