Method of treatment for dental implant to improve the osteointegration

Abstract

The present invention discloses a treatment for a dental implant, which improves its osteointegration.

Description

[0001] "Method of treatment for dental implant to improve the osteointegration"

[0002] DESCRIPTION

[0003] The present invention finds application in the field of dental implants and particularly concerns a method for improving the osteointegration of dental implants .

[0004] Background of the invention

[0005] Dental implant therapy is a well-established treatment in case of a missing or loss of a tooth . The osseointegration process allows the direct contact and growth of bone tissue on the implant surface . Long-term bone stability around the implant is affected maj orly by the healthiness of the supra- crestal soft tissue seal . The soft tissue seal is established in the first three months after the latest soft tissue wound around the implant neck . The tissue architecture is defined by different compartments moving from the bone to the mucosal margin : ( 1 ) a connective tissue collar of about 1- 1 . 5 mm; ( 2 ) the j unctional epithelium of about 1 . 0 mm; ( 3 ) a sulcular epithelium of 1 . 0 mm .

[0006] The long-term success of dental implants depends on the establishment and maintenance of crestal bone levels concerning the formation of a soft tissue barrier

[0001] . The peri-implant soft tissues have become a maj or focusing point as the presence of an effective mucosal attachment at the transmucosal part of the implant can provide the implant with the protection of the bone from bacteria contamination and prevent peri-implant pathologies [ 1-3 ] . This aspect is of particular relevance also in terms of the aesthetic treatment outcome long term, since the stability of the gingival margin and papilla is related to the maintenance of crestal bone

[0007]

[0003] . Cell adhesion at both soft and hard tissue levels is influenced by the surface properties of dental implants and implant intra-mucosal components or abutments. These properties include topography, roughness, chemistry, charge, and hydrophilicity [4-6] . Therefore, there has been a tendency to investigate methods to change surface chemistry to promote faster cell interaction at the bone and soft tissue levels as well as stability of the tissues once healed [7-10] .

[0008] Due to the lack of attachment of connective tissue fibers to the implant surface, implant collars have been modified including a microtextured portion to enhance soft tissue attachment to the implant cervical area [11,12] . On the other hand, some experimental and clinical studies have shown the physical attachment of the connective tissue to the laser microtextured collar of dental implants with less bone loss than smooth collars [9,13-15] .

[0009] Dental implant therapy is a well-established treatment in case of a missing or loss of a tooth. The osteointegration process allows the direct contact and growth of bone tissue on the implant surface. Long-term bone stability around the implant is affected majorly by the healthiness of the supra- crestal soft tissue seal. The soft tissue seal is established in the first three months after the latest soft tissue wound around the implant neck. The tissue architecture is defined by different compartments moving from the bone to the mucosal margin: (1) a connective tissue collar of about 1- 1.5 mm; (2) the junctional epithelium of about 1.0 mm; (3) a sulcular epithelium of 1.0 mm.

[0010] The long-term success of dental implants depends on the establishment and maintenance of crestal bone levels concerning the formation of a soft tissue barrier [1] . The peri-implant soft tissues have become a major focusing point as the presence of an effective mucosal attachment at the transmucosal part of the implant can provide the implant with the protection of the bone from bacteria contamination and prevent peri-implant pathologies [ 1-3 ] . This aspect is of particular relevance also in terms of the aesthetic treatment outcome long term, since the stability of the gingival margin and papilla is related to the maintenance of crestal bone

[0003] .

[0011] Cell adhesion at both soft and hard tissue levels is influenced by the surface properties of dental implants and implant intra-mucosal components or abutments . These properties include topography, roughness , chemistry, charge , and hydrophilicity [ 4- 6 ] . Therefore , there has been a tendency to investigate methods to change surface chemistry to promote faster cell interaction at the bone and soft tissue levels as well as stability of the tissues once healed [ 7-10 ] .

[0012] Due to the lack of attachment of connective tissue fibers to the implant surface , implant collars have been modified including a microtextured portion to enhance soft tissue attachment to the implant cervical area [ 11 , 12 ] . On the other hand, some experimental and clinical studies have shown the physical attachment of the connective tissue to the laser microtextured collar of dental implants with less bone loss than smooth collars [ 9 , 13-15 ] .

[0013] Despite the above there is still the need for a treatment that may allow an increased osteointegration between the collagen fibers of the connective tissue layer to the implant / abutment surface mediated by chemical interaction .

[0014] Summary of the invention The inventors of the present patent application have surprisingly found that a dental implant suitably treated shows an improved osteointegration and physiological engraftment thus forming a stable connective seal.

[0015] Object of the invention

[0016] In a first object the present invention discloses a method for the surface treatment of a dental implant to improve the osteointegration.

[0017] The obtained treated dental implants represent further objects of the invention .

[0018] In a second object the present invention discloses the use of a solution of phenolic compounds and of a solution of gelatin to improve the osteointegration of dental implants.

[0019] Brief description of the figures

[0020] Figure 1 - Scanning Electron Microscopy observation of untreated titanium samples .

[0021] Figure 2 - Scanning Electron Microscopy observation of polyphenols / gelatin-treated titanium samples.

[0022] Figure 3 - L929 cell viability onto scaffolds. L929 cells were seeded at a density of 8xl04cells and cell viability was measured at 24hs, 48hs, 72hs, and 7 days. (A-F) The survival rate of the different samples is the function of the time and is shown as histograms (A-C) and curve charts (D-F) , respectively. Cell viability was expressed as cell number (A, D) , survival rate as a percentage compared to NT (B, E) , and fold increase to seeded cells (C, F) . Results are presented as mean ± SD (n=3, symbols indicate statistical significance vs NT (*) , vs PO (#) ; *** / ### p < 0.001; ** / ## p < 0.01; * / # p < 0.05) . (G-I) Survival rate of the different samples in function of the materials and shown as histograms . Cell viability was expressed as cell number (G) , survival rate as a percentage compared to NT (H) , and fold increase to seeded cells (I) . Results are presented as mean ± SD (n=3, symbols indicate statistical significance vs 24 (*) , vs 48 (#) , vs 72h (§) ; *** / ### p < 0.001; ** / ## p < 0.01; * / # / § p < 0.05) .

[0023] Figure 4 - pFAK evaluation. CLSM images showing the expression of focal adhesion p-FAK (green, 488 Alexa Fluor) on plastic surfaces (CTRL positive) , NT, P0, and PG were acquired at 20* magnification. Nuclei were stained with Hoechst 33342 (blue) . Scale bars: 100 pm.

[0024] Figure 5 - Collagen I evaluation. A) CLSM images, showing the expression of type I collagen (green, 488 Alexa Fluor) on NT, P0, PG, and plastic surfaces were acquired at 20* magnification. Nuclei were stained with Hoechst 33342 (blue) . Scale bars: 100 pm. B) ELISA assay was performed on the three different scaffolds; data were shown as B) ng of collagen production per each scaffold or C) per cell respectively; D, E) the same data were compared to NT materials. n=3 ** p < 0.01; **p < 0.05.

[0025] Figure 6 - Quantification of bacterial adhesiveness evaluated on a Gram-positive strain of S. oralis on three different titanium samples: non-treated (NT) , polyphenols treated (PO) , and polyphenol / gelatin treated (PG) .

[0026] Detailed description of the invention

[0027] In a first object the present invention discloses a method for the surface treatment of a dental implant to improve the osteointegration .

[0028] According to the present invention, the method comprises the steps of :

[0029] I) providing a dental implant,

[0030] II) incubating said dental implant alternatively in a first solution and in a second solution, optionally, III) incubating said dental implant in the second solution .

[0031] For the purposes of the present invention, "dental implant" means a post inserted into the alveolar bone to support an artificial tooth or other prosthesis.

[0032] Usually, a dental implant is made of metal, such as titanium (or its alloys) and may have a screw shape (cylindrical or conical shaped) .

[0033] The dental implant could be based on a single piece or multiple pieces; nevertheless, one component is in the bone to obtain direct bone growth and contact (osteointegration) , a part concerns the soft tissue compartment (connective tissue, junctional epithelium, functional epithelium, and sulcular epithelium) and a part aims to support the prosthetic tooth or prosthesis.

[0034] For the purposes of the present invention, the dental implant that can be subjected to the disclosed method can be parallel or conically shaped .

[0035] For the purposes of the present invention, the surface of the dental implant may be resulting from a surface treatments, such as, for example: f luoride-modif ied surface, oxidized, acid-etched, machined, sandblasted, laser modified or combinations of them; or coatings, such as, for example: ceramics (hydroxyapatite, titania, diamond-like carbon, etc) , metals (porous titanium, etc) , or biomolecules (growth factors, etc) or composites (combing organic and / or inorganic materials) .

[0036] For the purposes of the present invention, dental implant comprises commercially available dental implants in native / pristine bone, grafted bone (simultaneously or healed / staged) , and post-extraction sites. For the purposes of the present invention, in step II) the first solution is a solution of phenolic compounds.

[0037] For the purposes of the present invention, "phenolic compounds" refers to molecules characterized, at least in part thereof, by the presence of an aromatic nucleus (benzene ring) bound to one or more hydroxyl functional groups .

[0038] Non-limitative examples of such compound include: simple phenols (molecules with a single benzene ring and containing only hydroxyl groups as substituents, e.g. phenol and hydroquinone) , phenolic aldehydes (containing both the phenolic group and the aldehyde group, e.g. vanillin) , phenolic acids (e.g. cinnamic acids) , phenylamines (amphoteric molecules containing a weakly acidic group and a strongly basic group, e.g. phenylalanine) , phenol compounds (the phenolic ring is bound to another benzene ring or to other heterocyclic compounds that have hydroxyl / lactone / ketone functional groups, e.g. coumarins and xanthones) , flavonoids (made up of two benzene rings connected by a chain with three carbon atoms that constitutes an oxygenated heterocyclic ring, e.g. catechins, flavonons, flavones, chaicones, flavanonols, flavanols, leucoanthocyanidin, anthocyanidin) , phenylpropanoids (characterized by the presence of an aromatic ring with an aliphatic side chain with three carbon atoms, es. hydroxycinnamic acids) and tannins. In the present description the terms "phenols" and "polyphenols" can have the same meaning and can be used together or as synonyms .

[0039] For the purposes of the present invention, the first solution may comprise one phenolic compound or a mixture thereof.

[0040] According to a preferred aspect of the invention, the phenolic compounds, polyphenolic compounds, or derivatives thereof for the treatment of a dental implant are represented by derivatives of cinnamic acid and tannin .

[0041] In particularly preferred aspect , the cinnamic acid derivatives are constituted by caffeic acid .

[0042] In particularly preferred aspect , the tannin derivatives are constituted by tannic acid .

[0043] According to a preferred aspect of the invention, the first solution comprises at least one phenyl derivative of cinnamic acid represented by caffeic acid .

[0044] In particular , caffeic acid may be in a concentration comprised between 1 mM and 1 M .

[0045] According to a preferred aspect of the invention, the first solution comprises at least one phenyl derivative of tannin represented by tannic acid .

[0046] In particular, tannic acid may be in a concentration comprised between 0 . 01 mM and 1 . 5 M .

[0047] According to a particularly preferred aspect of the invention, the first solution comprises caffeic acid and tannic acid (within the concentration above disclosed) .

[0048] According to a preferred aspect of the invention, the phenolic solution ( the first solution ) is a buffered solution .

[0049] Buffers may be represented by phosphate buffers or other suitable buffers .

[0050] According to a preferred aspect of the invention, the pH of the phenolic solution is comprised between 3 and 9 .

[0051] For the purposes of the present invention, in step II ) the incubation with the first solution is continued for a period of 1 to 60 minutes . For the purposes of the present invention, in step I I ) the second solution is a gelatin solution or a solution of collagen-based derivatives .

[0052] The term "collagen-based derivatives" refers to synthetic or natural matrices composed primarily of collagen .

[0053] Collagen is non-toxic and has minimal immunogenicity .

[0054] Collagen can be processed in several ways leading to the following derivatives : porous sponges , gels / gelatin, and sheets .

[0055] In particular , gels / gelatin is an irreversibly hydrolyzed form of collagen, wherein the hydrolysis reduces protein fibrils into smaller peptides ; depending on the physical and chemical methods of denaturation, the molecular weight of the peptides falls within a broad range .

[0056] Gelatin may also be referred to as hydrolyzed collagen, collagen hydrolysate , gelatin hydrolysate , hydrolyzed gelatin, and collagen peptides after it has undergone hydrolysis . Gels / gelatins can be available in various types ( gelling / non-gelling ) and obtained from various sources ( synthesis / porcine / bovine ) . Substances containing gelatin or functioning similarly are called gelatinous substances .

[0057] Collagen- and gelatin-based derivatives can be employed as it stands or functionalized with linkers to improve the bioactivity or the integration with the surrounded tissues .

[0058] Typical functionalization includes crosslinking with activated linkers or with bioactive molecules .

[0059] According to a preferred aspect of the invention, the gelatin solution is a buffered solution .

[0060] Buffers may be represented by phosphate buffers or other suitable buffers . According to a preferred aspect of the invention, the concentration of gelatin in the solution may be comprised between 0 . 05 mg / ml and 100 mg / ml .

[0061] According to a preferred aspect of the invention, the pH of the gelatin solution is the comprised between 5 and 10 .

[0062] For the purposes of the present invention, in step II ) the incubation with the second solution is continued for a period of 1 to 60 minutes .

[0063] According to a preferred aspect of the invention, step I I ) is performed at a temperature between 5 ° C and 95 ° C .

[0064] As per the method of the invention, before step I ) the dental implant may be subj ected to a pre-treatment step, wherein it is treated with isopropanol .

[0065] In particular, the dental implants are placed in isopropanol (preferably, pure isopropanol ) for a time between 1 and 60 minutes .

[0066] The samples are then washed from the isopropanol and subj ected to incubation in the phenolic solution ( the first solution ) .

[0067] In particular, there is carried out one incubation step of from 1 minute to 6 hours and preferably of 30 minutes in the first solution .

[0068] Each step is followed by a washing step with deionized water , preferably of 5 minutes .

[0069] According to the present invention, in step II ) the incubation is performed alternatively in the first solution and in the second solution .

[0070] According to a preferred embodiment of the invention, in step II ) there are performed an incubation step in the first solution for a time between 2 minutes and 6 hours , and preferably of about 30 minutes , followed by an incubation step in the second solution for a time between 1 minutes and 6 hours and preferably of about 15 minutes . A washing step is then performed in deionized water for a time between 2 minutes and 2 hours .

[0071] In a preferred aspect of the invention, the alternation of steps is repeated between 1 and 30 times .

[0072] According to an optional aspect of the invention, a step I II ) can be performed, wherein the dental implant treated according to step II ) are incubated in the second solution ( gelatin solution) .

[0073] All the steps are carried out keeping the samples in moderate but constant stirring at a temperature between 5 ° C and 95 ° C .

[0074] The dental implants treated according to the method above disclosed represent further obj ects of the invention .

[0075] In a second obj ect the present invention discloses the use of a solution of polyphenols and of a solution of gelatin for the treatment of dental implant .

[0076] According to a first aspect , such a use improves the osteointegration of dental implants .

[0077] According to a second aspect , such a use promotes the physiological engraftment of the implanted dental implant by forming a stable connective seal .

[0078] The term "Connective Seal" refers to the compartment based on connective tissue rich in collagen fibers covering the underlining bone and having a relationship with the intra-mucosal component of the dental implant . This area protects the bone from bacteria contamination and / or infection .

[0079] A stable connective seal is formed when patient mucosal cells colonize , differentiate and produce an adequate collagen network . According to a third aspect , such a use significantly inhibits the onset of infections after implant insertion, healing and / or loading .

[0080] As a consequence , the use above disclosed prevents and / or inhibits peri-implantitis and peri-implant mucositis .

[0081] Peri-implantitis is defined as the pathological condition around dental implants characterized by inflammation in the peri-implant mucosa, which may lead to the progressive bone loss .

[0082] Peri-implant mucositis is characterized by inflammation in the implant surrounding mucosa without concomitant bone loss .

[0083] Peri-implant diseases in general are considered analogous to gingivitis and periodontitis , with some differences in the host response .

[0084] According to the present invention, there is inhibited the infection from periodontal pathogens and opportunistic microorganisms , and particularly from S. aureus, Streptococcus anaerobusc and oralis, Escheri chia coll , Candida, and Streptococci spp .

[0085] Many studies have been conducted to compare the microbiological flora of periodontitis and peri-implantitis . The first step for the initiation of bacterial colonization is forming a salivary pellicle consisting of salivary proteins and peptides . When an implant is exposed to the oral cavity, this pellicle forms rapidly on its surface and mediates microorganisms ' adhesion . Taken collectively, current data confirm that peri-implant infections are dominated by gram-negative bacteria , similar to periodontal infections , but some cases may harbor a distinct microbiota . Although early reports showed similarities between the peri-implant and periodontal flora , later studies demonstrated that peri-implantitis lesions might present consensus periodontal pathogens and opportunistic microorganisms, such as S. aureus, Streptococcus anaerobusc and oralis, Escherichia coll, Candida, and Streptococci spp .

[0086] The invention is further disclosed in the following Experimental Section .

[0087] Scanning Electron Microscopy evaluation

[0088] To evaluate the presence of the gelatin / polyphenol coating some titanium samples were observed by SEM microscopy. Before the observations, all the samples were subjected to metallization with a gold / palladium sputtering as required by practice for this type of investigation. At high magnifications, no appreciable difference is observed between untreated and treated samples. By increasing the magnifications (80.000 - 120.000 X) it is possible to appreciate the presence of a superficial layer on the treated samples (Fig.2) not visible on the untreated counterpart (Fig. 1) •

[0089] In vitro evaluation of the viability, adhesiveness, and morphology of 1292 cells and their production of type I collagen on different types of titanium samples

[0090] Three different materials of titanium differently coated have been tested with L929 cells: a material without treatment (NT) , a material coated with polyphenols (P0) , and a material coated with polyphenols and gelatin (PG) .

[0091] CELL SEEDING CONDITIONS

[0092] Before cell seeding, all scaffolds were sterilized three times with EtOH 70% and washed with sterile Milliq water. Murine fibroblast L929 were cultured in Minimum essential medium with Earl' s BSS, 10% Horse serum, 1% glutamine, % sodium pyruvate, 1% non-essential amino acids, and 1% penicillin-streptomycin . Culture flas ks were incubated under standard culture conditions at 37 ° C with 5 % CO2 in a humidified incubator . In vitro cell culture studies were performed with mouse connective tissue fibroblasts L929 to investigate the biocompatibility of the provided materials . This cell line is commonly used in cytotoxicity tests and represents a standard model for biocompatibility testing [ 20-22 ] .

[0093] CELL VIABILITY

[0094] The cells directly seeded on the scaffolds were placed in 48-well tissue culture plates , and 8xl 04cells were seeded onto the top of each scaffold with 300 pl of complete medium and incubated at 37 ° C in a humidified atmosphere with 5% CO2 . After 24hs , the dis ks were transferred into new sterile 48 -well plates containing 300pl of fresh medium for 24hs , 48hs , 72hs , and 7 days . At each time point , the culture medium was replaced with 300 l of base medium with 10% Alamar Blue solution, and cell viability was measured at 600nm by a microplate reader (Clariostar instrument ) . A standard curve of cell viability was used to express the results as the number of cells . After rinsing with PBS and flooding with 300 pl of fresh culture medium, plates were replaced in the incubator . The medium was changed every 48 hs until the completion of the assay . In Figure 3 the data are represented in the function of times (panels 1A-1F ) and function of scaffold types (panels 1G-1I ) . The cell viability is represented as follows : number of total cells (panels A, D, G) ; percentage of cells in comparison with NT scaffolds (panels B , E , H ) ; fold increase of cells in comparison to the number of seeded cells (panels C , F, I ) . Considering the time dependence , the number of cells on PG was higher in all time points concerning the other two samples (panels A, and D) . These data were confirmed considering the percentage of cells in comparison to NT (panels B, E) . In function of scaffold types, for each sample was detected a greater growth on PG and NT whereas on PO was reduced over time (panels G-I) . The data expressed as fold increase of cells (panels C, F, I) revealed that the cells showed the highest growth on each type of scaffold between 24hs and 48hs ( ****p<0.0001 ) .

[0095] DOT BLOT

[0096] After 24hs, cells were scraped from all samples and lysed with ice-cold lysis buffer (RIPA buffer lx containing 1 mM and lx protease inhibitor, Protease Inhibitor Tablets) for 30 min on ice. The lysates were evaluated with the BCA Protein Assay Kit to perform dot blot analyses .

[0097] After the sample was dried, primary antibodies anti-FAK (diluted 1:1000) , anti-phosphorylated FAK (pY397) (diluted 1:1000) , and appropriate secondary HRP-conj ugated antibodies were used. Detection was performed with Western Chemiluminescent HRP substrate and revealed using an ImageQuant LAS4000 imaging system Densitometry analysis was carried out with Imaged software.

[0098] ELISA ASSAY

[0099] The deposition of Type I Collagen and Fibronectin was evaluated at 7 days by ELISA assay as previously described

[0023] . Briefly, to evaluate the amount of the protein on the scaffold surface, the samples were washed extensively with sterile PBS to remove the culture medium, and then incubated for 24 hs at -20°C with 300 pl of sterile sample buffer (20 mM Tris-HCl, 4 M GuHCl, lOmM EDTA, 0,066% [w / v] SDS, pH 8.0) . At the end of the incubation period, total protein concentration was evaluated with the BCA Protein Assay Kit and then an ELISA assay was performed. In brief, to measure the amount of each protein by ELISA, wells were coated with pg of extract protein, in coating buffer (50 mM Na2COs, pH 9.5) overnight at 4°C. Control wells were coated with bovine serum albumin (BSA) as a negative control. After three washes with PBS containing 0.1% (v / v) Tween20, the wells were blocked by incubating with BSA 2% for Ih at RT . The wells were subsequently incubated for Ih at RT with anti-Type-I collagen (1:500 in 2% BSA) and anti-f ibronectin polyclonal antisera (50 ng / ml in 2% BSA) . After washing, the wells were incubated for Ih at RT with horseradish peroxidase (HRP) -conjugated goat anti-rabbit IgG (1:2000 dilution in 2% BSA) , and the signal was revealed with SigmaFast OPD. The absorbance reading was performed at 450 nm.

[0100] CONFOCAL LASER SCANNING MICROSCOPY ANALYSIS (CLSM)

[0101] A cell suspension of 8xl04cells was added onto the top of each scaffold and on the cell culture coverslip and incubated at 37°C in a humidified atmosphere with 5% CO2. After 24 hs, samples were washed with 1*PBS, fixed with 4% (w / v) paraformaldehyde solution (PFA) for 30 min at 4°C, washed with phosphate-buff ered saline (PBS) three times for 5min, and then treated as described in the following sections.

[0102] Focal adhesion studies

[0103] The focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase that distinctly co-localizes with integrin at sites of attachment to their ligands. It binds directly or indirectly the cytoplasmic domains of integrin and constitutes a bridge between integrin and cytoskeleton, which produces signal transduction inside the cells able to adhere to the surfaces

[0024] . Attachment, adhesion, and spreading are typical processes of the first phase of cell-material interactions

[0025] . For focal adhesion analysis, paraformaldehyde-fixed cells were permeabilized with 0.1% Triton X-100 for 5 min at room temperature (RT) . Then L929 cells were further incubated with primary mouse anti-p-FAK (pY397) (1:250 in 1% BSA) overnight. Afterward, samples were incubated with a specific secondary antibody for immunofluorescence observation at a concentration of 1:1.500 in 1% BSA. Hoechst 33342 (2 pg / mL) was used for nuclei staining. The images were taken using a confocal microscope (Leica Microsystems, Bensheim, Germany) equipped with a digital image capture system at 20* magnification .

[0104] After 24 hs of cells seeding on the scaffolds, the activation focal adhesion kinase was evaluated using both qualitative and quantitative assays. In Figure 4, a different scenario was observed on the three different types of scaffolds. Cells seeded on both scaffolds, NT and P0, displayed a rounder shape, and the adhesion process seems to be at an early stage (pFAK presence was low) . On the contrary, on the PG scaffold, the seeded cells showed protrusions and exhibited a network of pFAK evidencing the cell attempt to form contacts with the fibrous architecture of the scaffold surface. These data confirm the previous viability results, indicating that the PG scaffold seems to promote cell adhesion concerning the other two scaffold types.

[0105] Collagen and fibronectin deposition

[0106] For Collagen type I and Fibronectin deposition on the scaffold analysis, paraformaldehyde-fixed cells were incubated with primary rabbit anti-Coll I and anti-f ibronectin polyclonal antisera (1:500 in 1% BSA) overnight. Afterward, samples were incubated with specific secondary antibodies for immunofluorescence, used at a concentration of 1:1.500 in 1% BSA. Hoechst 33342 (2pg / mL) was used for nuclei staining. The images were taken using a confocal microscope (Leica Microsystems, Bensheim, Germany) equipped with a digital image capture system at 20 xmagnif ication

[0026] .

[0107] Collagen is an abundant structural protein in all animals and is the most prevalent component of the extracellular matrix ( ECM) ( 8 ) . On day 7 , the CLSM images showed that the cultured L929 was able to deposit type I collagen differently on the three scaffolds . In Figure 5A, NT and PG scaffolds seem to be qualitatively similar in terms of collagen deposition, whereas PO showed the lowest value of green fluorescence . These data were confirmed by ELISA quantitative assay ( Fig . 5B-5E ) . Data were shown as ng of collagen per each scaffold (panels B and D) or ng of collagen per cell (panels C and E ) , respectively . A significant increase in collagen type I deposition was observed on the PG samples compared to the PO scaffolds both in terms of collagen per scaffold (panels B and D) and collagen per cell (panels C and E ) .

[0108] Evaluation test of adhesiveness on chirurgical titanium samples

[0109] The test has been performed to evaluate the contribution of polyphenols / gelatin coating on the microbial adhesiveness in treated surfaces of surgical titanium. Samples were provided as discs , ready to be tested . Three sets of samples were provided, and treated in three different ways : without treatment (NT ) , a material coated with polyphenols ( PO ) , and a material coated with polyphenols and gelatin ( PG ) . The analytical activity consists of testing the adhesiveness of a bacteria strain of Streptococcus oralis . In particular, the test provides for an incubation of the specimen with a specific microbial strain at a known concentration . At the end of the incubation time with the strain, samples are slightly rinsed to remove microorganisms leaning or loosely adhered on the surface then a vigorous rinse passage is aimed to detach microorganisms strongly adhered to the specimen . All the rinsing liquids are analyzed and seeded in agar plates and incubated at specific conditions and temperatures according to the growth requirements of the microorganism.

[0110] Every test has a triplicate sample for each of the three treatments , a triplicate sample without treatment , a positive control consisting of cotton fabric, and a negative control for each of the three treatments and for the untreated sample as a specimen not incubated with the microorganism.

[0111] Results are expressed as a percentage decrease in bacterial growth . The microorganism used for the test was Streptococcus orali s ATCC 6249 .

[0112] Preparation of the inoculum - The test uses a quantified microbial suspension in MRD buffer . Suspension is quantified by cell count at the microscope .

[0113] Contamination of samples - The analysis is performed in triplicate , i . e . the same conditions are tested 3 times for samples . All test samples are immersed in a 10 ml suspension of Streptococcus orali s in MRD buffer at a concentration of about 10 5 UFC / ml , in 60mm sterile Petri dishes for 90 minutes , in a shaking plate at 100 rpm speed .

[0114] Seeding on agar plates - At the end of the incubation time , each test sample and each control is transferred into a new 60mm sterile Petri dish containing 10 ml of MRD . The plates are mixed gently for 30 seconds . The MRD content of each Petri dish is seeded in sterile 90 mm Petri dishes in agar medium, with spread plate technique , after dilution in MRD broth . This step is called "Rinse" .

[0115] After this , each test sample and each control is placed inside a tube containing 10 ml of MRD and they are subj ected to 3 vortex cycles at a maximum speed of 3 minutes each . The MRD content of each tube is seeded in sterile 90 mm Petri dishes in a specific agar medium, with a spread plate technique , after dilution in MRD broth . This step is called "Vortex" .

[0116] Table 1 shows the result of the quantification of bacterial adhesiveness evaluated on a Gram-positive strain of S . orali s on three different titanium samples : non-treated (NT ) , polyphenols treated ( PO ) , and polyphenol / gelatin treated ( PG ) . The percentage of bacterial reduction was quantified by counting the colony forming units (CPU) determined after different washing steps ( rinse and vortex ) by comparison with positive control ( n=3 for each type of sample ) .

[0117] From what is reported in Table 1 , it is clear that untreated titanium is a material that is autonomously refractory to the bacterial adhesiveness of a strain of S. orali s (NT samples ) . The polyphenolic mixture guarantees the maintenance of this characteristic ( PO samples ) . Noteworthy, the use of gelatins for the formulation of culture media for cells and bacteria is linked to the nutritional and mechanical supportive properties that this substance can provide . Despite its presence in the titanium samples ( PG) , the percentage of anti-adhesive effect compared to the control is not significantly different from that of the untreated samples or those treated with polyphenols alone ( PO ) . By linking the results of the test with the bacterial strain and those of the cell growth with the Murine fibroblast L929 it is possible to confirm that the presence of the collagen component helps to improve cell homing and metabolism activation but at the same time inhibits bacterial attachment and proliferation . Conclusi ons

[0118] The evidences above reported show that the treatment of the invention promotes the cellular colonization and the production of extracellular matrix , while inhibiting the bacterial colonization of a Gram-positive bacterial strain such as the S . oralis . In view of the above description, the advantages of the present invention will be apparent to the person skilled in the art .

[0119] The method of the present invention can be applied to titanium surfaces that may be subj ected to surface treatments such as fluoride- modified surface , oxidized, acid-etched, machined, sandblasted, laser modified, or combinations thereof which can be used for manufacturing dental implant .

[0120] The method of the present invention can be applied to dental implants in native / pristine bone , grafted bone ( simultaneously or healed / staged ) , and post-extraction sites .

[0121] The method of the present invention can be applied to dental implants in metals (porous titanium, etc ) , biomolecules ( growth factors , etc ) or composites ( combing organic and / or inorganic materials ) .

[0122] The method of the invention can be carried out with conventional devices and machines , so that it can be promptly integrated within the production lines already existing .

[0123] In addition to the above technical advantages , the method of the invention significantly inhibits , after implant insertion, healing , and / or loading, the onset of infections .

[0124] Peri-implant infections are dominated by gram-negative bacteria , similar to periodontal infections , but some cases may harbor a distinct microbiota . Although early reports showed similarities between the periimplant and periodontal flora , later studies demonstrated that peri- implantitis lesions might present consensus periodontal pathogens and opportunistic microorganisms , such as S. aureus, Streptococcus anaerobusc and oralis, Escheri chia coll , Candida, and Streptococci spp . REFERENCES

[0125] 1. Welander M et al. Clin. Oral Implants Res. 2008;19:635-641.

[0126] 2. Berglundh T et al. Clin. Oral Implants Res. 2007;18:1-8.

[0127] 3. Berglundh T et al. J. Clin. Periodontol. 1996;23:971-973.

[0128] 4. Zhao G et al. J. Biomed. Mater. Res. Part A 2005;74:49-58.

[0129] 5. Rompen E et al. Clin. Oral Implants Res. 2006;17:55-67.

[0130] 6. Wennerberg A et al. Clin. Oral Implants Res. 2009;20:172-184.

[0131] 7. Cosyn J et al. J. Periodontol. 2007;78:1657-1663.

[0132] 8. Goene RJ et al. Int. J. Periodontics Restor. Dent. 2007;27:211-219.

[0133] 9. Nevins M et al. Int. J. Periodontics Restor. Dent. 2010;30:245-255.

[0134] 10. Sul YT et al. J. Biomed. Mater. Res. Part A 2009;89:942-950.

[0135] 11. Moon IS et al. J. Clin. Periodontol. 1999;26:658-663.

[0136] 12. Nevins M et al. Int. J. Periodontics Restor. Dent. 2008;28:111-121.

[0137] 13. Nevins M et al. Int. J. Periodontics Restor. Dent. 2012;32:385-392.

[0138] 14. Nevins ML et al. Int. J. Periodontics Restor. Dent. 2012;32:497-507.

[0139] 15. Shapoff CA et al. Int. J. Periodontics Restor. Dent. 2010;30:129-137.

[0140] 16. Sawase T et al. Clin. Oral Implants Res. 2008;19:491-496.

[0141] 17. Tugulu S et al. J. Mater. Sci. Mater. Med. 2010;21:2751-2763.

[0142] 18. Duske K et al. J. Clin. Periodontol. 2012;39:400-407.

[0143] 19. Tomasi C et al. Clin. Oral Impl. Res. 25 ; 2014 : 997-1003.

[0144] 20. Lasocka I et al. Toxicol In Vitro. 2018;48:276-285.

[0145] 21. Guidetti G et al. Polymer Degradation and Stability 2020; 182 : 109403.

[0146] 22. Karakecili AG et al. J Biosci Bioeng. 2007 ; 104 ( 1 ) : 69-77.

[0147] 23. Saino E et al. Eur Cell Mater. 2011;21:59-72; discussion 72.

[0148] 24. Khalili AA et al. Int J Mol Sci. 2015;16(8) :18149-84.

[0149] 25. Mitra SK et al. Nat Rev Mol Cell Biol. 2005;6(l) :56-68.

[0150] 26. Martelli G et al. Biomacromolecules. 2020; 21 (3) : 1157-1170.

[0151] 27. Shoulders MD et al. Annu Rev Biochem. 2009;78:929-958.

Claims

CLAIMS1. A method for the treatment of a dental implant to improve the osteointegration comprising the steps of:I) providing a dental implant,II) incubating said dental implant alternatively in a first solution, which is a phenolic solution, and in a second solution, which is a gelatin solution or a solution of collagen-based derivatives .

2. The method for the treatment of a dental implant to improve the osteointegration according to the preceding claim, wherein in step II) the phenolic solution comprises a phenolic compound selected from the group comprising: simple phenols, which are molecules with a single benzene ring and containing only hydroxyl groups as substituents, e.g. phenol and hydroquinone, phenolic aldehydes, which are molecules containing both the phenolic group and the aldehyde group, e.g. vanillin, phenolic acids, such as cinnamic acids, phenylamines, such asamphoteric molecules containing a weakly acidic group and a strongly basic group, e.g. phenylalanine, phenol compounds wherein the phenolic ring is bound to another benzene ring or to other heterocyclic compounds that have hydroxyl / lactone / ketone functional groups, e.g. coumarins and xanthones, flavonoids made up of two benzene rings connected by a chain with three carbon atoms that constitutes an oxygenated heterocyclic ring, e.g. catechins, flavonons, flavones, chaicones, flavanonols, flavanols, leucoanthocyanidin, anthocyanidin, phenylpropanoids characterized by the presence of an aromatic ring with an aliphatic side chain with three carbon atoms, such as hydroxycinnamic acids and tannins; or a mixture thereof.3 . The method for the treatment of a dental implant to improve the osteointegration according to the preceding claim, wherein in step I I ) the phenolic solution comprises derivatives of cinnamic acid and derivatives of tannin .4 . The method for the treatment of a dental implant to improve the osteointegration according to the preceding claim, wherein in step I I ) the phenolic solution comprises caffeic acid and tannic acid .5 . The method for the treatment of a dental implant to improve the osteointegration according to the preceding claim, wherein in step I I ) the phenolic solution comprises caffeic acid in a concentration comprised between 1 mM and 1 M .6 . The method for the treatment of a dental implant to improve the osteointegration according to the preceding claim, wherein in step I I ) the phenolic solution comprises tannic acid in a concentration comprised between 0 . 01 mM and 1 . 5 M .7 . The method for the treatment of a dental implant to improve the osteointegration according to any one of the preceding claims , wherein in step I I ) each incubation is performed at a temperature of between 5 ° C and 95 ° C .8 . The method for the treatment of a dental implant to improve the osteointegration according to any one of the preceding claims , further comprising a step I II ) incubating said dental implant in the second solution, which is a gelatin solution or a solution of collagen-based derivatives .

9. The method for the treatment of a dental implant to improve the osteointegration according to any one of the preceding claims , whereinthe alternate incubation with the first solution and the incubation with the second solution are repeated for 1 to 30 times .10 . The method for the treatment of a dental implant to improve the osteointegration according to any one of the preceding claims , wherein each one of the incubation with the first solution and the incubation with the second solution is continued for 1 minute to 6 hours .11 . The method for the treatment of a dental implant to improve the osteointegration according to any one of the preceding claims , further comprising a pre-treatment step with isopropanol followed by a washing step .12 . The method for the treatment of a dental implant to improve the osteointegration according to the preceding claim, wherein after the pretreatment step with isopropanol , the dental implant is incubated with the first solution, which is a phenolic solution, followed by a washing step .

13. The method for the treatment of a dental implant to improve the osteointegration according to the preceding claim, wherein said pretreatment step of incubation with the first solution is continued for 1 minute to 6 hours and preferably for about 30 minutes .14 . A dental implant obtained according to the method of any one of the preceding claims .

15. The use of a solution of polyphenols and of a solution of gelatin for the treatment of dental implant .

16. The use of a solution of polyphenols and of a solution of gelatin according to the preceding claim to improve the osteointegration of dental implants .17 . The use of a solution of polyphenols and of a solution of gelatin according to the preceding claim 15 or 16 to promote the physiological engraftment of the implanted dental implant by forming a stable connective seal .18 . The use of a solution of polyphenols and of a solution of gelatin according to any one of the preceding claims 15 to 17 to inhibit the onset of infections after implant insertion, healing and / or loading .

19. The use of a solution of polyphenols and of a solution of gelatin according to the preceding claim to prevent and / or inhibit peri-implantitis and peri-implant mucositis .20 . The use of a solution of polyphenols and of a solution of gelatin according to the preceding claim to prevent and / or inhibit peri-implantitis and peri-implant mucositis caused by S. aureus, Streptococcus anaerobusc and oralis, Escheri chia coll , Candida, and Streptococci spp .

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