Final Report Summary - NANOTI (Development of a titanium dental implant with superior antibacterial properties)
Executive Summary:
Dental implants are medical devices that are placed into the jawbone to support restorations that resemble a tooth or a group of teeth. In the last couple decades antibiotic resistance has triggered an urgent problem also in this field, mainly due to the overuse of antimicrobial medicines. As a result, the incidence of infection-associated destructive inflammatory diseases has significantly increased in the last decade. Such negative processes affect both soft and hard tissues surrounding dental implants which are supposedly triggered by multiple bacteria colonies making complete recovery very hard to achieve, even by the combination of surgical decontamination and targeted antibiotic therapy.
Several biomaterials have been developed recently as potential implant materials and lots of distinguished scientific achievements have been translated into implant products. However, the improved biocompatibility of implant materials with human tissue entails their increased susceptibility for bacterial infections as well. Bacterial biofilm formation on the surface of dental implants poses severe threat as bacteria concentrated in biofilms of are significantly more resistant to antibiotics, rendering such treatment ineffective.
Currently available solutions focus on antimicrobial-releasing coatings enabling high local concentration of antibiotics as a preventive therapy. However, due to the limited drug reservoir capacity these solutions provide only a temporary protection in the first few days.
Within the NANOTI project a long-lasting nanostructured surface with unique anti-microbial properties has been developed enabling to reduce affinity for bio-film formation in long-term.
Scientific background: The surface properties of an implant determine its interactions with the surrounding host tissue. Physicochemical and mechanical properties of the surface, like wettability, surface free energy and surface topography, are of prime importance for the optimization of adhesion, spreading and proliferation of cells. The initial response when material is placed in the biological surroundings is the water molecule adsorption to its surface. This happens within the first few nanoseconds. In the second stage the protein adsorption occurs. In general, it can be stated that small proteins will be the first to adsorb due to their rapid transport to the surface. Over time, bigger ones that have greater affinity to the surface replace these proteins. Wettability, more precisely, surface free energy of the substrate is known to influence protein adsorption. It is usually reported that biomaterial surface with moderate hydrophilicity improved cell growth and biocompatibility. However, cell adhesion can decrease as the material becomes very hydrophilic. This points out to the existence of a range of optimal surface energies. The third stage of biological response includes the cell attachment to the surface. This stage is influenced by adsorbed protein layer as well as surface topography. Cell spreading and differentiation are known to be influenced especially by both micro-, and nanoscale roughness and wettability.
Utilising above discussed know-how the NANOTI project developed novel nanophase topographies with special surface properties unfavourable for bacterial adhesion and biofilm formation, while the bone integration properties were shown to be better or equivalent to state-of-the-art sandblasted and etched surfaces.
The developed nanostructure is based on the latest scientific findings and results without adding any antibacterial compound. In total 5 different surface states have been developed. Together with the novel nanosurfaces also the associated surface treatment procedure has been developed. The NANOTI project also focused on process scale-up research and the development of a prototype of industrial scale semi-automated surface treatment equipment. Special attention was paid to adapt the surface treatment process to the complex geometry of dental implants. With the final version of the equipment and the treatment process the consortium could successfully achieve uniform coverage and surface quality on dental implants. The current prototype of the treatment system would be able to process 5000 implants per month considering a two shift operation. The treatment system prototype allows varying the surface treatment properties in a wide range, enabling the SMEs to continue experimental research fur further improvement of the surface properties.
The second half of the project concentrated on the biological validation of the newly developed surface developed by means of in-vitro and in-vivo studies. Based on the in-vitro studies, as well as other destructive and non-distractive investigations two surfaces types have been selected as most promising, the nanotubular and the nanopitted surfaces. The biological performance of the first is slightly better, while later performs better concerning chemical and mechanical stability. The NANOTI surfaces in general 1) showed significantly reduced biofilm formation, 2) while showing equally good osseointegration (bone healing around the dental implant) performance, relative to the commercially used surfaces.
The nanopitted surface was also evaluated by the (clinic and distributor) end user partners as more attractive. Its gold like shine gives the implant a real premium appearance. The SME partner plan to proceed with a post-project clinical validation of the NANOTI coated implants and plan in parallel to utilize the collected know-how for further improvement of the NANOTI surface.
Project Context and Objectives:
Dental implants are medical devices that are placed into the jawbone to support restorations that resemble a tooth or a group of teeth. In the last couple decades antibiotic resistance has triggered an urgent problem also in this field, mainly due to the overuse of antimicrobial medicines. As a result, the incidence of infection-associated destructive inflammatory diseases has significantly increased in the last decade. Such negative processes affect both soft and hard tissues surrounding dental implants which are supposedly triggered by multiple bacteria colonies making complete recovery very hard to achieve, even by the combination of surgical decontamination and targeted antibiotic therapy.
Several biomaterials have been developed recently as potential implant materials and lots of distinguished scientific achievements have been translated into implant products. However, the improved biocompatibility of implant materials with human tissue entails their increased susceptibility for bacterial infections as well. Bacterial biofilm formation on the surface of dental implants poses severe threat as bacteria concentrated in biofilms of are significantly more resistant to antibiotics, rendering such treatment ineffective. In some cases the locally low doses may even contribute to development of resistance.
Surface modification with the aim to discourage biofilm formation on implants seems to be a promising solution in order to counteract biofilm formation on the implant surface. Currently available solutions focus on antimicrobial-releasing coatings enabling high local concentration of antibiotics as a preventive therapy. However, due to the limited drug reservoir capacity these solutions provide only a temporary protection in the first few days. The aim of this project is to develop a long-lasting surface with unique anti-microbial properties enabling to reduce affinity for bio-film formation in long-term.
The latest scientific results suggest that nanostructured surfaces exhibit antibacterial properties while enhance the bone healing around the implant. NANOTI project aimed to develop novel nanophase topography with special surface properties unfavorable for bacterial adhesion and biofilm formation. The developed nanostructure is based on the latest scientific findings and results without adding any antibacterial compound. NANOTI aims to produce a surface coating 1) showing significantly reduced biofilm formation, 2) while showing equally good osseointegration (bone healing around the dental implant) performance, relative to the commercially used surfaces.
Research and development objectives of NANOTI focused on three main elements: 1) development of the proper surface treatment procedure, 2) development of an automated surface treatment equipment prototype and 3) biological validation of the surface developed by means of in-vitro and in-vivo studies.
Project Results:
The surface treatment process developed in NANOTI project is based on the sequence of individual chemical and electrochemical steps. The first development period was focusing on the phasing and optimization of the process parameters with the aim to produce surface states that meet the target requirements. The performance of the surface treatment process was characterized in non-destructive mechanical and in-vitro microbiology tests. For this reason during the research phase the surface treatment processes were optimized to titanium discs instead of 3-dimensional screw-like implants. The 2-dimensional plane of the titanium discs allowed the comparable quantitate analysis of the surfaces and the quantification of bacterial adhesion.
In the initial research run stable nanotubular TiO2 arrays have been successfully created on titanium discs and it was successfully demonstrated that pathogenic bacteria were less prone to attach than on the commercially available implant surfaces. In a next step the surface treatment process was adapted to the 3-dimensional screw-like implants.
The surface treatment procedure comprises three separate steps, out of which electrochemical polishing is the first stage, which aims to produce a smooth, mirror-like surface. The second surface modification method involved in the project is chemical etching to prepare the surface for the growth of nanostructured titanium oxide. The third surface treatment method is anodic oxidation which creates the desired nanosurfaces by converting the balk titanium surface to structured titanium oxide. The pattern and surface energy of the created nanosurfaces have key roles in the behavior of the implants in microbiology and biocompatibility tests and these the properties depend mainly on the applied anodizing conditions, such as composition of the electrolyte, treatment time, voltage and current characteristics, stirring velocity as well as and cathode material can significantly influence the morphology, structure and chemical composition of the created TiO2 layer.
Beside the process development, the project also aimed to scale-up the treatment process and to develop a semi-automated surface treatment equipment prototype allowing to treat multiple implants in parallel in a reproducible manner. An industrial desk serves as a supportive unit for the electrolyte tanks and for other auxiliary units. Special important holders have been developed with integrated cathode as well as mechanical support and electrical connection to the implants. The electrolyte circulation system was implemented in a way to provide as far as possible the same conditions at each treated implant to avoid any unwanted cross influence between parallel treated implants, and the reduce thermal bobble formation on the surface of the anode.
An electrical power supply and control system has been developed which can be flexibly set in a wide parameter range to support experimental research. The electronics box provides the high power output for electro polishing and anodization process and it controls all supportive systems such as pump, mixer, heater and cooling units. The PLC based control system features an easy-to-use touch screen controlled graphical user interface enabling to run automated profiles with exact timings. The control software also provides options for treatment profile adjustment, data logging as well as manual operation.
Several surface investigation methods have been used to determine the relevant properties of the developed NANOTI surfaces. Scanning electron microscopy was used to visualize the resulting nanostructures on the surface, and among others surface roughness, wettability and surface free energy has been measured as most relevant parameters.
Distractive investigations were applied to determine the mechanical integrity and failure modes of the anodic films. Mechanical strength and stability were investigated by scratch and screwing test methods to determine the damage resistance of the oxide-substrate system. Chemical stability was investigated by a corrosion resistance test in aqueous chloride media in acidic environment, which is important to determine biocompatibility and long-term stability of the anodic layer.
The nanotubular type anodic film showed the highest wettability and surface free energy on the discs. In contrast, nano-pitted type anodic film showed in comparison a slightly lower wettability and surface free energy, while the state-of-the-art sandblasted/etched surface showed a quite hydrophobic property. However, the wettability values showed remarkably lower standard deviation in case of nano-pitted anodic film compared to nanotubular film. The confocal microscopic measurements revealed differences in the profile roughness of the anodic films (nanoporous: Ra = 0,74±0,1, Rz = 3,92±0,58; nano-pitted: Ra = 0,75±0,11, Rz = 3,58±0,29) in contrast to the electropolished surface (Ra = 0,15±0,05, Rz = 0,7±0,23) that may be correlated with the different biological features.
However, the nanoporous anodic films showed unfortunately in the scratch and screwing tests low damage resistance and proneness to exfoliation that may limit its applicability in praxis. In contrast, the additionally developed nano-pitted anodic film showed considerably higher mechanical stability with even less material extrusion than the blank electro polished surface. Similarly the results of the corrosion tests showed also better chemical stability in case of the nanopitted surface type in contrast to nanotubular type.
The bacterial adhesion properties were investigated in supporting in-vitro studies. First biofilm formation was investigated under static conditions, than further bacterial adhesion experiments were performed under dynamic conditions as an attempt to simulate the saliva flow in the human oral cavity. Two different fluid velocities representative for stimulated (5.8 mm/s) and unstimulated (0.1 mm/s) saliva flow were tested. In all cases that ‘gold standard’ sandblasted/etched titanium oxide surface showed the highest remaining bacterial coverage, while the developed NANOTI surfaces showed considerably less coverage comparable to the completely flat electropolished surface. While in the static test the nanotubular surfaces performed the best, in the dynamic adhesion tests the nanopitted surfaces showed the least amount of bacterial coverage. By increasing the flow rate of the culture medium the amount of bacteria remaining on the substrate surface decreased significantly.
On the other hand, the consortium also assessed the influence of the surface type on cell adhesion, proliferation, and osteogenic differentiation of human mesenchymal stem cells (hMSC) for the two most promising surface types: a nanoporous and nanopitted, while using electropolished as neutral reference. Sandblasted/acid etched samples served as a positive control as they constitute the current gold standard of titanium implant surfaces. Proliferation and differentiation was followed over a period of 35 days, and differentiation studies included the osteogenic marker alkaline phosphatase (ALP) on the protein expression level as well as the quantification of mineralization based on cellular deposition of calcium phosphate in the extracellular matrix. The osteogenic differentiation was additionally analysed by measuring the expression of genes typically associated with the osteogenic phenotype, and the production of pro-inflammatory cytokines (PGE-2, IL 6 and IL8) by the hMSC was investigated. All experiments were performed with human bone marrow stromal cells from two different donors.
Cells adhered well on all surfaces, but on both NANOTI surfaces they exhibited a well spread morphology with well-developed stress fibers and with only small differences in focal adhesion between the polished and nanostructured surfaces. Cells on the sandblasted reference exhibited the most obvious differences in morphoplogy with an elongated and spindle-like shape. Cell proliferation, on the other hand, was significantly higher on nanopitted surfaces compared to all other samples, and significantly lowest on the reference sandblasted surface. Alkaline phosphatase activity was highest for cells on nanotubular surface, followed by sandblaseted reference, while for polished and nanopitted it was somewhat lower. Calcium phosphate deposition showed a similar characteristic.
Interleukine and PEG2 secretion was comparatively low for all surfaces, but the lowest values were obtained for cells on nanotubular surfaces. Osteogenic differentiation as measured by expression of ALP, RUNX2, Coll1 and BSP also indicated that the nanotubular surfaces state may be the most supportive for osteogenic differentiation out of the investigated surface types.
The nanotubular was the one with the most intense nanotexturing, and this most strongly affected the differentiation behavior and lead to a more osteogenic phenotype. It performed similar or better (especially with respect to cell proliferation and mineral deposition) than the current gold standard SBAE, which made it the most promising from osseointegration point of view for developments in the field of surface modifications for bone applications. The nanopitted surface state appeared to promote osteogenic differentiation to slightly less but still useful degree, making it also feasible for hard tissue applications. Considering all critical results the nanopitted surface can be considered as the best performing compromise for clinical application for dental implants where high mechanical stability is also of key importance.
The project concluded with an animal study. The goal of the animal study was to investigate the osseo-integration of the implants and in parallel to study the susceptibility to reduce peri-implant infections to extrapolate to the expected performance in humans. In the animal study, many incidental unwanted effects might be uncovered that exceed the limitations of in vitro studies, like aseptic inflammation triggered by the generation of wear particles or the involvement of the immune system.
Up to now, the preliminary results of the in vivo osseo-integration study does not allow for a concluding discussion. Hence, it is required to wait for the evaluation of the histological stains.
Potential Impact:
Potential impact:
The solution that NanoTi technology could offer has been elaborated to a specific problem: the increasing number of infection after the planting of dental implants. The reason why the number of the infected patients increasing is that, on the first hand replacing missing teeth with implants are getting even more common and affordable. On the second hand, the number of dental implant manufacturer companies on the market is also rising, so the quality and price range of the products on the market became quiet wide. Thanks to these two factors, the average quality of the dental implants became lower than it was a couple of years before. Based on our research, the average potential infection rate by newly planted implants in the EU27 is 15-20%, which means, that out of the 7 million implants placed in patients in the EU every year, about 1,05-1,2 million has a high chance to be removed because of peri-implantitis. The goal of our project is, to decrease the infection with a ratio of 50%! The reason of this is simple: Serious cases of peri-implantitis result in several visits to a dentist for surgical treatment and to remove and replace the infected implant, making the patient unable to work for at least 5 days due to the visits, surgery and required time for healing.
To represent NanoTi’s potential in numbers (annual):
- save up to 27.000 days of sick-leave
- save up to 3.140.000 EUR potential loss occurred of sick-leave
- save up to ~100-150 EUR per piece of infected implants (est.: 68.750.000 EUR)
Related to the budget of the project, and the necessary amount for further investment, the numbers above are very significant and clearly showing the impact of the project both economic and social point of view.
Main dissemination activities:
The consortium acknowledged that communicating the importance of research and development and its contribution to economic competitiveness is an essential dimension of achieving project objectives but also an opportunity to increase the visibility for European companies in dental implantology.
In light of the project relevance the main dissemination objectives were to raise awareness on implementing an optimal biocompatible nanosurface to reduce the risk of dental implant-associated infections, to provide and insight on the project’s solutions, to provide appropriate information to parties in the industrial, academical and public levels, to promote the results to potential customers and third parties and to provide evidence based findings such as publications, testing and pilot production.
The main stakeholders of the project were scientific and research communities, dentists, implantologists, manufacturers, policy makers, dental patients and the general public. To deliver NanoTi’s messages, the partners of the consortium used different dissemination platforms and instruments. The main channels of dissemination were:
-project website
-digital media
-scientific and business articles on popular websites
-market relevant project presentations
-workshops (both internal and external)
-business meetings
-direct mailing
-trade fairs
-exhibitions
-industry events
-networking events
-scientific publications
-conferences
Based on the key established aspects of dissemination the consortium has completed number of promotion activities:
▪ Creation of a NanoTi brand strategy to become a substantial player amongst implant surface technologies.
▪ Project logo: Partners have selected a project logo that is widely used in documents promoting the project and its results.
▪ Project flyer and poster: a PDF flyer for print and one for e-mailing have been drafted that can be used in conferences and other events. Both materials contain the details of the partnership, the description of the problem to be solved and the innovative results developed. Partners can download the printable formats of the flyer from the project website for their convenient use at dissemination occasions. Partners plan to translate the flyers and posters along with other general dissemination materials into their languages to enhance the impact of the project at a national level.
▪ Project website: One of the most important dissemination channels of the project is the website that contains regularly updated information for all stakeholders. The website was set up after project start and was developed continuously during the lifetime of the project.
▪ Information on NanoTi was included on the websites of project partners and NanoTi also appeared on some thematic websites.
▪ Newsletter: four issues were prepared by the Consortium and distributed among possible future customers.
▪ Manuscripts: An article in English has been prepared that describes the general objectives and introduces the project partners.
▪ Press release: Articles have been created and published on the websites of Business Quarter and the University of Birmingham.
▪ Innovation Conference (13. 02. 2014) organized by Ateknea Solutions in Budapest, Hungary: The event centered on the topic of “H2020” was attended by more than 100 SMEs and EU professionals. As part of the conference program Ateknea introduced its portfolio of ongoing and recently completed research projects including the NanoTi initiative. Several bilateral and group discussions took place where information on the project was shared. The event and interviews with the organizers were covered by the local media.
▪ Annual Innovation Day (23.02.2014) in Budapest, Hungary organized by Eötvös Loránd University (ELTE) and the Europe Direct information office: The participants represented institutes with a research and development profile as well as government agencies and the core of the scientific life on a national level. This event is part of the efforts to encourage the utilization of knowledge generated by the academia and put the results to practical use and to foster industry-academia partnerships. Ateknea exhibited on the innovation day and spread information on its project portfolio including NanoTi.
▪ July 2014-CMOSET 2014, Grenoble, France (Invited Talk, N.E. Vrana): “How to Make Implants and Tissue Engineering Products Smarter? Inclusion of Biosensors and Methods to Control and Adjust Host Response Real-time.” PROTIP was invited to present their work at the NanoBioTechnology Symposium of CMOS Emerging Technologies Research. The potential use of anodisation for infection prevention of the implants was presented with the mention of NanoTi.
▪ E. Rieger, G. Koenig, P. Schultz, A. Dupret-Bories, C. Debry, D. Vautier, P. Lavalle, N.E. Vrana 2014. Titanium Microbead Based Implant Architectures: From Single Beads to Complex 3D Structures. French-German-Polish Symposium of Architectured Biomaterials, Medical and Tissue Engineering, Berlin, Germany (Poster Presentation) 4-5 December 2014 Audience: Scientists, Ambassadors, Embassy Science Officers, Audience size: 150-200, Language: English
▪ In addition to these presentations PROTIP had a full-time stand for 3 days in April 2014 at the European Laryngology Society Annual Meeting 2014 in Antalya, Turkey. During this event PROTIP presented their products and their ongoing research projects including NanoTi. PROTIP had one-to-one meetings with Head and Neck surgeons, medical device distributors where the biofilm formation and Candida Albicans growth was pointed out as possible problems for PROTIP products. Here PROTIP has explained the goals of NanoTi and how it will be applicable to the titanium based implants in the Head and neck field. Audience: 500, composed of medical students, professors and surgeons, otorhinolaryngology related companies. Language: English.
▪ ITM disseminated the project at the University of Birmingham during the British Science Week (8-13 Sept). The UoB is a strategic partner of ITM in the post-project development phase. There were several events during the course of the week and numerous valuable contacts were made from the scientific community and from the industry as well.
▪ E. Rieger, A. Dupret-Bories, L. Salou, M. Metz-Boutigue, P. Layrolle, C. Debry, P. Lavalle, N.E. Vrana 2015. Controlled implant/soft tissue interaction by nanoscale surface modifications of 3D porous titanium implants, Nanoscale, Impact factor: 7.1
Exploitation of results:
The exploitable results include (1) an electrochemical surface treatment procedure encompassed in (2) a machine that is suitable for creating (3) nanotubular and nano-pitted TiO2 arrays on dental implants (and with slight modifications other titanium implants) that reduce the risk of implant-associated infections. These results constitute the tangible output of an integrated technology platform developed in the NanoTi project, whose commercial exploitability relies either on the industrial applicability of the machine or on the licensing of the surface treatment method as a know-how. The scientific hypotheses and technological assumptions addressed in the NanoTi project are reduced to practice i) by the surface treatment machine and manifested ii) in nanotubular and nano-pitted anodic films that show unique biological features. This means that outputs of the NanoTi project are standalone technological solutions that are separable that provide the independent accessories of a novel surface treatment procedure.
To protect the results of the project, first of all an in-depth patent infringement search has been carried out (both in the EU and US), which has been periodically updated during the project. The outcome of the so called freedom to operate search was, that there are not any patent documents existing, which could be enforceable against our desired file. Based on the results, project partner ITM submitted two patents: a PCT and a National application.
List of Websites:
www.nanoti.eu
Dental implants are medical devices that are placed into the jawbone to support restorations that resemble a tooth or a group of teeth. In the last couple decades antibiotic resistance has triggered an urgent problem also in this field, mainly due to the overuse of antimicrobial medicines. As a result, the incidence of infection-associated destructive inflammatory diseases has significantly increased in the last decade. Such negative processes affect both soft and hard tissues surrounding dental implants which are supposedly triggered by multiple bacteria colonies making complete recovery very hard to achieve, even by the combination of surgical decontamination and targeted antibiotic therapy.
Several biomaterials have been developed recently as potential implant materials and lots of distinguished scientific achievements have been translated into implant products. However, the improved biocompatibility of implant materials with human tissue entails their increased susceptibility for bacterial infections as well. Bacterial biofilm formation on the surface of dental implants poses severe threat as bacteria concentrated in biofilms of are significantly more resistant to antibiotics, rendering such treatment ineffective.
Currently available solutions focus on antimicrobial-releasing coatings enabling high local concentration of antibiotics as a preventive therapy. However, due to the limited drug reservoir capacity these solutions provide only a temporary protection in the first few days.
Within the NANOTI project a long-lasting nanostructured surface with unique anti-microbial properties has been developed enabling to reduce affinity for bio-film formation in long-term.
Scientific background: The surface properties of an implant determine its interactions with the surrounding host tissue. Physicochemical and mechanical properties of the surface, like wettability, surface free energy and surface topography, are of prime importance for the optimization of adhesion, spreading and proliferation of cells. The initial response when material is placed in the biological surroundings is the water molecule adsorption to its surface. This happens within the first few nanoseconds. In the second stage the protein adsorption occurs. In general, it can be stated that small proteins will be the first to adsorb due to their rapid transport to the surface. Over time, bigger ones that have greater affinity to the surface replace these proteins. Wettability, more precisely, surface free energy of the substrate is known to influence protein adsorption. It is usually reported that biomaterial surface with moderate hydrophilicity improved cell growth and biocompatibility. However, cell adhesion can decrease as the material becomes very hydrophilic. This points out to the existence of a range of optimal surface energies. The third stage of biological response includes the cell attachment to the surface. This stage is influenced by adsorbed protein layer as well as surface topography. Cell spreading and differentiation are known to be influenced especially by both micro-, and nanoscale roughness and wettability.
Utilising above discussed know-how the NANOTI project developed novel nanophase topographies with special surface properties unfavourable for bacterial adhesion and biofilm formation, while the bone integration properties were shown to be better or equivalent to state-of-the-art sandblasted and etched surfaces.
The developed nanostructure is based on the latest scientific findings and results without adding any antibacterial compound. In total 5 different surface states have been developed. Together with the novel nanosurfaces also the associated surface treatment procedure has been developed. The NANOTI project also focused on process scale-up research and the development of a prototype of industrial scale semi-automated surface treatment equipment. Special attention was paid to adapt the surface treatment process to the complex geometry of dental implants. With the final version of the equipment and the treatment process the consortium could successfully achieve uniform coverage and surface quality on dental implants. The current prototype of the treatment system would be able to process 5000 implants per month considering a two shift operation. The treatment system prototype allows varying the surface treatment properties in a wide range, enabling the SMEs to continue experimental research fur further improvement of the surface properties.
The second half of the project concentrated on the biological validation of the newly developed surface developed by means of in-vitro and in-vivo studies. Based on the in-vitro studies, as well as other destructive and non-distractive investigations two surfaces types have been selected as most promising, the nanotubular and the nanopitted surfaces. The biological performance of the first is slightly better, while later performs better concerning chemical and mechanical stability. The NANOTI surfaces in general 1) showed significantly reduced biofilm formation, 2) while showing equally good osseointegration (bone healing around the dental implant) performance, relative to the commercially used surfaces.
The nanopitted surface was also evaluated by the (clinic and distributor) end user partners as more attractive. Its gold like shine gives the implant a real premium appearance. The SME partner plan to proceed with a post-project clinical validation of the NANOTI coated implants and plan in parallel to utilize the collected know-how for further improvement of the NANOTI surface.
Project Context and Objectives:
Dental implants are medical devices that are placed into the jawbone to support restorations that resemble a tooth or a group of teeth. In the last couple decades antibiotic resistance has triggered an urgent problem also in this field, mainly due to the overuse of antimicrobial medicines. As a result, the incidence of infection-associated destructive inflammatory diseases has significantly increased in the last decade. Such negative processes affect both soft and hard tissues surrounding dental implants which are supposedly triggered by multiple bacteria colonies making complete recovery very hard to achieve, even by the combination of surgical decontamination and targeted antibiotic therapy.
Several biomaterials have been developed recently as potential implant materials and lots of distinguished scientific achievements have been translated into implant products. However, the improved biocompatibility of implant materials with human tissue entails their increased susceptibility for bacterial infections as well. Bacterial biofilm formation on the surface of dental implants poses severe threat as bacteria concentrated in biofilms of are significantly more resistant to antibiotics, rendering such treatment ineffective. In some cases the locally low doses may even contribute to development of resistance.
Surface modification with the aim to discourage biofilm formation on implants seems to be a promising solution in order to counteract biofilm formation on the implant surface. Currently available solutions focus on antimicrobial-releasing coatings enabling high local concentration of antibiotics as a preventive therapy. However, due to the limited drug reservoir capacity these solutions provide only a temporary protection in the first few days. The aim of this project is to develop a long-lasting surface with unique anti-microbial properties enabling to reduce affinity for bio-film formation in long-term.
The latest scientific results suggest that nanostructured surfaces exhibit antibacterial properties while enhance the bone healing around the implant. NANOTI project aimed to develop novel nanophase topography with special surface properties unfavorable for bacterial adhesion and biofilm formation. The developed nanostructure is based on the latest scientific findings and results without adding any antibacterial compound. NANOTI aims to produce a surface coating 1) showing significantly reduced biofilm formation, 2) while showing equally good osseointegration (bone healing around the dental implant) performance, relative to the commercially used surfaces.
Research and development objectives of NANOTI focused on three main elements: 1) development of the proper surface treatment procedure, 2) development of an automated surface treatment equipment prototype and 3) biological validation of the surface developed by means of in-vitro and in-vivo studies.
Project Results:
The surface treatment process developed in NANOTI project is based on the sequence of individual chemical and electrochemical steps. The first development period was focusing on the phasing and optimization of the process parameters with the aim to produce surface states that meet the target requirements. The performance of the surface treatment process was characterized in non-destructive mechanical and in-vitro microbiology tests. For this reason during the research phase the surface treatment processes were optimized to titanium discs instead of 3-dimensional screw-like implants. The 2-dimensional plane of the titanium discs allowed the comparable quantitate analysis of the surfaces and the quantification of bacterial adhesion.
In the initial research run stable nanotubular TiO2 arrays have been successfully created on titanium discs and it was successfully demonstrated that pathogenic bacteria were less prone to attach than on the commercially available implant surfaces. In a next step the surface treatment process was adapted to the 3-dimensional screw-like implants.
The surface treatment procedure comprises three separate steps, out of which electrochemical polishing is the first stage, which aims to produce a smooth, mirror-like surface. The second surface modification method involved in the project is chemical etching to prepare the surface for the growth of nanostructured titanium oxide. The third surface treatment method is anodic oxidation which creates the desired nanosurfaces by converting the balk titanium surface to structured titanium oxide. The pattern and surface energy of the created nanosurfaces have key roles in the behavior of the implants in microbiology and biocompatibility tests and these the properties depend mainly on the applied anodizing conditions, such as composition of the electrolyte, treatment time, voltage and current characteristics, stirring velocity as well as and cathode material can significantly influence the morphology, structure and chemical composition of the created TiO2 layer.
Beside the process development, the project also aimed to scale-up the treatment process and to develop a semi-automated surface treatment equipment prototype allowing to treat multiple implants in parallel in a reproducible manner. An industrial desk serves as a supportive unit for the electrolyte tanks and for other auxiliary units. Special important holders have been developed with integrated cathode as well as mechanical support and electrical connection to the implants. The electrolyte circulation system was implemented in a way to provide as far as possible the same conditions at each treated implant to avoid any unwanted cross influence between parallel treated implants, and the reduce thermal bobble formation on the surface of the anode.
An electrical power supply and control system has been developed which can be flexibly set in a wide parameter range to support experimental research. The electronics box provides the high power output for electro polishing and anodization process and it controls all supportive systems such as pump, mixer, heater and cooling units. The PLC based control system features an easy-to-use touch screen controlled graphical user interface enabling to run automated profiles with exact timings. The control software also provides options for treatment profile adjustment, data logging as well as manual operation.
Several surface investigation methods have been used to determine the relevant properties of the developed NANOTI surfaces. Scanning electron microscopy was used to visualize the resulting nanostructures on the surface, and among others surface roughness, wettability and surface free energy has been measured as most relevant parameters.
Distractive investigations were applied to determine the mechanical integrity and failure modes of the anodic films. Mechanical strength and stability were investigated by scratch and screwing test methods to determine the damage resistance of the oxide-substrate system. Chemical stability was investigated by a corrosion resistance test in aqueous chloride media in acidic environment, which is important to determine biocompatibility and long-term stability of the anodic layer.
The nanotubular type anodic film showed the highest wettability and surface free energy on the discs. In contrast, nano-pitted type anodic film showed in comparison a slightly lower wettability and surface free energy, while the state-of-the-art sandblasted/etched surface showed a quite hydrophobic property. However, the wettability values showed remarkably lower standard deviation in case of nano-pitted anodic film compared to nanotubular film. The confocal microscopic measurements revealed differences in the profile roughness of the anodic films (nanoporous: Ra = 0,74±0,1, Rz = 3,92±0,58; nano-pitted: Ra = 0,75±0,11, Rz = 3,58±0,29) in contrast to the electropolished surface (Ra = 0,15±0,05, Rz = 0,7±0,23) that may be correlated with the different biological features.
However, the nanoporous anodic films showed unfortunately in the scratch and screwing tests low damage resistance and proneness to exfoliation that may limit its applicability in praxis. In contrast, the additionally developed nano-pitted anodic film showed considerably higher mechanical stability with even less material extrusion than the blank electro polished surface. Similarly the results of the corrosion tests showed also better chemical stability in case of the nanopitted surface type in contrast to nanotubular type.
The bacterial adhesion properties were investigated in supporting in-vitro studies. First biofilm formation was investigated under static conditions, than further bacterial adhesion experiments were performed under dynamic conditions as an attempt to simulate the saliva flow in the human oral cavity. Two different fluid velocities representative for stimulated (5.8 mm/s) and unstimulated (0.1 mm/s) saliva flow were tested. In all cases that ‘gold standard’ sandblasted/etched titanium oxide surface showed the highest remaining bacterial coverage, while the developed NANOTI surfaces showed considerably less coverage comparable to the completely flat electropolished surface. While in the static test the nanotubular surfaces performed the best, in the dynamic adhesion tests the nanopitted surfaces showed the least amount of bacterial coverage. By increasing the flow rate of the culture medium the amount of bacteria remaining on the substrate surface decreased significantly.
On the other hand, the consortium also assessed the influence of the surface type on cell adhesion, proliferation, and osteogenic differentiation of human mesenchymal stem cells (hMSC) for the two most promising surface types: a nanoporous and nanopitted, while using electropolished as neutral reference. Sandblasted/acid etched samples served as a positive control as they constitute the current gold standard of titanium implant surfaces. Proliferation and differentiation was followed over a period of 35 days, and differentiation studies included the osteogenic marker alkaline phosphatase (ALP) on the protein expression level as well as the quantification of mineralization based on cellular deposition of calcium phosphate in the extracellular matrix. The osteogenic differentiation was additionally analysed by measuring the expression of genes typically associated with the osteogenic phenotype, and the production of pro-inflammatory cytokines (PGE-2, IL 6 and IL8) by the hMSC was investigated. All experiments were performed with human bone marrow stromal cells from two different donors.
Cells adhered well on all surfaces, but on both NANOTI surfaces they exhibited a well spread morphology with well-developed stress fibers and with only small differences in focal adhesion between the polished and nanostructured surfaces. Cells on the sandblasted reference exhibited the most obvious differences in morphoplogy with an elongated and spindle-like shape. Cell proliferation, on the other hand, was significantly higher on nanopitted surfaces compared to all other samples, and significantly lowest on the reference sandblasted surface. Alkaline phosphatase activity was highest for cells on nanotubular surface, followed by sandblaseted reference, while for polished and nanopitted it was somewhat lower. Calcium phosphate deposition showed a similar characteristic.
Interleukine and PEG2 secretion was comparatively low for all surfaces, but the lowest values were obtained for cells on nanotubular surfaces. Osteogenic differentiation as measured by expression of ALP, RUNX2, Coll1 and BSP also indicated that the nanotubular surfaces state may be the most supportive for osteogenic differentiation out of the investigated surface types.
The nanotubular was the one with the most intense nanotexturing, and this most strongly affected the differentiation behavior and lead to a more osteogenic phenotype. It performed similar or better (especially with respect to cell proliferation and mineral deposition) than the current gold standard SBAE, which made it the most promising from osseointegration point of view for developments in the field of surface modifications for bone applications. The nanopitted surface state appeared to promote osteogenic differentiation to slightly less but still useful degree, making it also feasible for hard tissue applications. Considering all critical results the nanopitted surface can be considered as the best performing compromise for clinical application for dental implants where high mechanical stability is also of key importance.
The project concluded with an animal study. The goal of the animal study was to investigate the osseo-integration of the implants and in parallel to study the susceptibility to reduce peri-implant infections to extrapolate to the expected performance in humans. In the animal study, many incidental unwanted effects might be uncovered that exceed the limitations of in vitro studies, like aseptic inflammation triggered by the generation of wear particles or the involvement of the immune system.
Up to now, the preliminary results of the in vivo osseo-integration study does not allow for a concluding discussion. Hence, it is required to wait for the evaluation of the histological stains.
Potential Impact:
Potential impact:
The solution that NanoTi technology could offer has been elaborated to a specific problem: the increasing number of infection after the planting of dental implants. The reason why the number of the infected patients increasing is that, on the first hand replacing missing teeth with implants are getting even more common and affordable. On the second hand, the number of dental implant manufacturer companies on the market is also rising, so the quality and price range of the products on the market became quiet wide. Thanks to these two factors, the average quality of the dental implants became lower than it was a couple of years before. Based on our research, the average potential infection rate by newly planted implants in the EU27 is 15-20%, which means, that out of the 7 million implants placed in patients in the EU every year, about 1,05-1,2 million has a high chance to be removed because of peri-implantitis. The goal of our project is, to decrease the infection with a ratio of 50%! The reason of this is simple: Serious cases of peri-implantitis result in several visits to a dentist for surgical treatment and to remove and replace the infected implant, making the patient unable to work for at least 5 days due to the visits, surgery and required time for healing.
To represent NanoTi’s potential in numbers (annual):
- save up to 27.000 days of sick-leave
- save up to 3.140.000 EUR potential loss occurred of sick-leave
- save up to ~100-150 EUR per piece of infected implants (est.: 68.750.000 EUR)
Related to the budget of the project, and the necessary amount for further investment, the numbers above are very significant and clearly showing the impact of the project both economic and social point of view.
Main dissemination activities:
The consortium acknowledged that communicating the importance of research and development and its contribution to economic competitiveness is an essential dimension of achieving project objectives but also an opportunity to increase the visibility for European companies in dental implantology.
In light of the project relevance the main dissemination objectives were to raise awareness on implementing an optimal biocompatible nanosurface to reduce the risk of dental implant-associated infections, to provide and insight on the project’s solutions, to provide appropriate information to parties in the industrial, academical and public levels, to promote the results to potential customers and third parties and to provide evidence based findings such as publications, testing and pilot production.
The main stakeholders of the project were scientific and research communities, dentists, implantologists, manufacturers, policy makers, dental patients and the general public. To deliver NanoTi’s messages, the partners of the consortium used different dissemination platforms and instruments. The main channels of dissemination were:
-project website
-digital media
-scientific and business articles on popular websites
-market relevant project presentations
-workshops (both internal and external)
-business meetings
-direct mailing
-trade fairs
-exhibitions
-industry events
-networking events
-scientific publications
-conferences
Based on the key established aspects of dissemination the consortium has completed number of promotion activities:
▪ Creation of a NanoTi brand strategy to become a substantial player amongst implant surface technologies.
▪ Project logo: Partners have selected a project logo that is widely used in documents promoting the project and its results.
▪ Project flyer and poster: a PDF flyer for print and one for e-mailing have been drafted that can be used in conferences and other events. Both materials contain the details of the partnership, the description of the problem to be solved and the innovative results developed. Partners can download the printable formats of the flyer from the project website for their convenient use at dissemination occasions. Partners plan to translate the flyers and posters along with other general dissemination materials into their languages to enhance the impact of the project at a national level.
▪ Project website: One of the most important dissemination channels of the project is the website that contains regularly updated information for all stakeholders. The website was set up after project start and was developed continuously during the lifetime of the project.
▪ Information on NanoTi was included on the websites of project partners and NanoTi also appeared on some thematic websites.
▪ Newsletter: four issues were prepared by the Consortium and distributed among possible future customers.
▪ Manuscripts: An article in English has been prepared that describes the general objectives and introduces the project partners.
▪ Press release: Articles have been created and published on the websites of Business Quarter and the University of Birmingham.
▪ Innovation Conference (13. 02. 2014) organized by Ateknea Solutions in Budapest, Hungary: The event centered on the topic of “H2020” was attended by more than 100 SMEs and EU professionals. As part of the conference program Ateknea introduced its portfolio of ongoing and recently completed research projects including the NanoTi initiative. Several bilateral and group discussions took place where information on the project was shared. The event and interviews with the organizers were covered by the local media.
▪ Annual Innovation Day (23.02.2014) in Budapest, Hungary organized by Eötvös Loránd University (ELTE) and the Europe Direct information office: The participants represented institutes with a research and development profile as well as government agencies and the core of the scientific life on a national level. This event is part of the efforts to encourage the utilization of knowledge generated by the academia and put the results to practical use and to foster industry-academia partnerships. Ateknea exhibited on the innovation day and spread information on its project portfolio including NanoTi.
▪ July 2014-CMOSET 2014, Grenoble, France (Invited Talk, N.E. Vrana): “How to Make Implants and Tissue Engineering Products Smarter? Inclusion of Biosensors and Methods to Control and Adjust Host Response Real-time.” PROTIP was invited to present their work at the NanoBioTechnology Symposium of CMOS Emerging Technologies Research. The potential use of anodisation for infection prevention of the implants was presented with the mention of NanoTi.
▪ E. Rieger, G. Koenig, P. Schultz, A. Dupret-Bories, C. Debry, D. Vautier, P. Lavalle, N.E. Vrana 2014. Titanium Microbead Based Implant Architectures: From Single Beads to Complex 3D Structures. French-German-Polish Symposium of Architectured Biomaterials, Medical and Tissue Engineering, Berlin, Germany (Poster Presentation) 4-5 December 2014 Audience: Scientists, Ambassadors, Embassy Science Officers, Audience size: 150-200, Language: English
▪ In addition to these presentations PROTIP had a full-time stand for 3 days in April 2014 at the European Laryngology Society Annual Meeting 2014 in Antalya, Turkey. During this event PROTIP presented their products and their ongoing research projects including NanoTi. PROTIP had one-to-one meetings with Head and Neck surgeons, medical device distributors where the biofilm formation and Candida Albicans growth was pointed out as possible problems for PROTIP products. Here PROTIP has explained the goals of NanoTi and how it will be applicable to the titanium based implants in the Head and neck field. Audience: 500, composed of medical students, professors and surgeons, otorhinolaryngology related companies. Language: English.
▪ ITM disseminated the project at the University of Birmingham during the British Science Week (8-13 Sept). The UoB is a strategic partner of ITM in the post-project development phase. There were several events during the course of the week and numerous valuable contacts were made from the scientific community and from the industry as well.
▪ E. Rieger, A. Dupret-Bories, L. Salou, M. Metz-Boutigue, P. Layrolle, C. Debry, P. Lavalle, N.E. Vrana 2015. Controlled implant/soft tissue interaction by nanoscale surface modifications of 3D porous titanium implants, Nanoscale, Impact factor: 7.1
Exploitation of results:
The exploitable results include (1) an electrochemical surface treatment procedure encompassed in (2) a machine that is suitable for creating (3) nanotubular and nano-pitted TiO2 arrays on dental implants (and with slight modifications other titanium implants) that reduce the risk of implant-associated infections. These results constitute the tangible output of an integrated technology platform developed in the NanoTi project, whose commercial exploitability relies either on the industrial applicability of the machine or on the licensing of the surface treatment method as a know-how. The scientific hypotheses and technological assumptions addressed in the NanoTi project are reduced to practice i) by the surface treatment machine and manifested ii) in nanotubular and nano-pitted anodic films that show unique biological features. This means that outputs of the NanoTi project are standalone technological solutions that are separable that provide the independent accessories of a novel surface treatment procedure.
To protect the results of the project, first of all an in-depth patent infringement search has been carried out (both in the EU and US), which has been periodically updated during the project. The outcome of the so called freedom to operate search was, that there are not any patent documents existing, which could be enforceable against our desired file. Based on the results, project partner ITM submitted two patents: a PCT and a National application.
List of Websites:
www.nanoti.eu