Biofilms Research Center for Biointerfaces

Transcription

1 BIO FILMS INTERFACES Biofilms Research Center for Biointerfaces Biofilms Research Center for Biointerfaces A translational research programme at Malmö University Funded by the Knowledge foundation Progress report #6 January 1 st, 2010 December 31 st, 2010

2 Cover illustration Biofilms develop spontaneously on a surface in contact with a liquid phase containing biomolecules and micro-organisms. The initial phase in the development is rapid adsorption of surface active molecules, notably macromolecules such as proteins, to the surface forming an initial conditioning film. Next step is attachment of microorganisms. These organisms grow and interact with molecules from the liquid phase, cell produced matrix, and other organisms in the formation of a biological film (biofilm). Organisms and molecules within the biofilm possess unique characteristics not observed for the same species suspended or dissolved in the associated liquid phase. Contact information Biofilms Research Center for Biointerfaces Faculty of Health and Society Malmö University SE MALMÖ, Sweden Center director: Assoc. Prof. Johan Engblom (JE) Tel: +46-(0) (JE); +46-(0) (Adm. Coordinator Eva Nilsson) johan.engblom@mah.se Visiting adress: Skåne University Hospital, SUS (Entrance 49) MALMÖ

3 1 List of Center Members During the period of report the Center has comprised the following members (permanent staff, postdocs, PhD-students and technical and administrative staff). 1.1 Permanent staff Johan Engblom, Assoc Prof., Director Thomas Arnebrant, Prof., Director , Vice Director 2010 Tautgirdas Ruzgas, Prof. Gunilla Nordin-Fredrikson, Prof. (also part time LU) Per Ståhle von Schwerin, Prof. Ann Wennerberg, Prof. Gunnel Svensäter, Prof. Ali Massih, Prof. Håkan Ericsson, Assoc. Prof. Vitaly Kocherbitov, Assoc. Prof. Liselott Lindh, Assoc. Prof. Anette Gjörloff-Wingren, Assoc. Prof. Zoltan Blum, Assoc. Prof. Liu-Ying Wei, Assoc. Prof. Julia Davies, Assoc. Prof. Bertil Kinnby, Assoc. Prof. Christina Bjerkén, Assoc. Prof. Claes Wickström, Assoc. Prof. Lars Ohlsson, Dr Tove Sandberg, Dr. Maria Stollenwerk, Dr. Sergey Shleev, Dr. Anna Ketelsen, Dr. 1.2 Junior researchers and postdocs Olof Svensson, Dr. Olga Santos, Dr. Laura Varas, Dr. Jessica Neilands, Dr. Luis Chavez de Paz, Dr. Javier Sotres, Dr Ida Svendsen, Dr Peter Nilsson, Dr Alejandro Barrantes, Dr Jovice Bon Singh Ng, Dr Ryo Jimbo, Dr Viktor Andoralov, Dr 1.3 PhD students Jildiz Hamit Eminovski Ulf Hejman Alma Masic 3

4 Maria Pihl Sebastian Björklund (enrolled at LU) Anton Fagerström Yana Znamenskaya Adnan Safdar Christian Alfredsson Kindblom Marjan Dorkhan Kostas Bougas Magnus Falk Peter Lamberg Cathrine Albér Mariko Hayashi Lory Melin Svanborg Tuerdi Maimaitiyili 1.4 Technical and administrative staff Eva Nilsson, Administrative coordinator Ulrika Troedsson, Technician Agnethe Henriksson, Technician Madeleine Blomqvist, Technician Lina Pedersen, Technician 1.5 MSc students BMMT Master at HS Joynul Abedin Ameena Daftani Endale Asmare Hailu Rakibul Islam Lutfor Islam Abu Sayeed Khan Jabed Khandaker Shadi Movahed Bashiri Shifa Saleem Selva Kumar Subramanian Mohammed B Sunmonu Inger Anne Tveit Surendra Vutti Shaheen Mohammad Syful Islam BMMT Master at HS Haddel Ali Shoker Joy Chia Ihab Dahi Payam Delfani Susanna Tarasco Mohammad Zahir Uddin Petra Wicktor BMMT Master at HS Aseel Albayati Sheima Sultan Kadir Eleonora Dahlquist Rula Bahran Peter Lamberg Marianne Mårtensson Patrik Bauer MS Master at HS & TS Maihemutijiang Maimaiti Carl Mikaelsson Ajigul Nuermaimaiti Oyetunji Oladele Kazeem Wureguli Reheman Christian Ukoha Oji Erik Öberg Simayijiang Zhayida In addtition to these two year Master programmes, the Faculty of Odontology (OD) offers the Dentistry programme which is a continuous five year program to Master level. 4

5 1.6 Management and boards Biofilms Research Center for Biointerfaces is managed on a daily basis by the Center director and an Executive group, constituted by the heads of the individual research groups. The Steering group constitutes a link to central Malmö University, and involves the Deputy Vice-Chancellor and the Deans of the three faculties/schools involved in the research activities of the Center. The Reference group is an advisory board to the Director and contains representatives from Industry, Medicon Valley Alliance, Malmö University and other universities. Executive group Johan Engblom, Assoc. Prof., Director & Chairman for Thomas Arnebrant, Prof., Director & Chairman for Per Ståhle von Schwerin, Prof. Gunnel Svensäter, Prof. Ann Wennerberg, Prof. Gunilla Nordin-Fredrikson, Prof. Håkan Eriksson, Assoc. Prof. Steering group Eva Engquist, Deputy Vice-Chancellor, Malmö University, Chairman Margareta Östman, Prof., Dean Faculty of Health and Society Naser Eftekharian, Head School of Technology Malmö University Lars Bondemark, Prof., Dean Faculty of Odontology Reference group Martin Malmsten, Prof., Uppsala University, Chairman Ian Hamilton, Prof. em., University of Manitoba, Canada Peter Nordström, Senior Project Manager, Medicon Valley Alliance Yngve Sommarin, Dr., R&D Manager Euro-Diagnostica/Wieslab AB Magnus Christensson, Dr., R&D Manager AnoxKaldnes AB Markus Johnsson, Dr., Senior Director Pharm. Development Camurus AB Eva Engquist, Deputy Vice-Chancellor, Malmö University Zoltan Blum, Assoc. Prof., Malmö University 5

6 2 The Director s Report Biofilms - Research Center for Biointerfaces that has been established as a vital asset for Malmö University, is well reputed and carries a strong trademark within the university. Also, an increased regional awareness of the center s activities, including other academic institutions, industry and public sector, can be noted. Industry partners comment that the center has positioned Malmö University as an attractive partner and that it is an enterprise that benefits both industry and academia. During the period the center has expanded to twice its original size, now comprising 38 projects and 32 industry partners. Two strong research areas have evolved from the center activities, now established as two of the current eight research profiles at the university, i.e. Oral health and Biointerfaces. External research funding for the entire university amounts to 107 MSEK in 2010, where Biofilms Research Center for Biointerfaces contributes with 20%. Regarding output in terms of international journal publications the center share is 23% of the total university production Collaboration with external parties is a corner stone in the university strategy and the center is often put forward as a good example. Viktoria Fröjd, Helena Tassidis (enrolled at LU) and Rickard Hägglund (enrolled at LTH) successfully defended their PhD-theses in 2010, as did Ulf Hejman and Adnan Safdar defending their licentiate theses. Claes Wickström and Christina Bjerkén were accepted as Associate Professors and Sergey Shleev has pending applications as Associate Professor (Malmö) and Habil. Doctor of Science (Russia). Drs Olga Santos and Olof Svensson left their positions as researchers during the year, while five new post docs and five PhD students (three at Malmö University and two enrolled at Lund University) were recruited. Two PhD-students had research stays at partner sites, Alma Masic at University of Guelph University, Canada, and Anton Fagerström at AkzoNobel Surface Chemistry AB, Stenungsund, Sweden. Several senior researchers also payed visits to universities outside Sweden (e.g. China, Russia, Lithuania, Germany and UK). During 2010 we have produced 59(67) publications in international journals, 5 book chapters and 3 proceeding papers. Publications are in journals such as Trends in Immunology (impact factor 8.8), Atherosclerosis, Trombosis and Vascular Biology (7.5) Journal of Controlled Release (6.0), Biofouling (4.4), Langmuir (3.9), Journal of Endodontics (3.0), Clinical Oral Implant Research (2.9), International Journal of Solids and Structure (1.8) and Mathematical Biosciences (1.3). High impact factors indicate substantial scientific quality of the research carried out within the Center. In addition to this, approximately 51 oral and 21 poster presentations were made at national and international meetings. In reviewing the number citations over the past five years on papers produced by the Center members we sum up 7763 for permanent staff and 587 for junior researchers and post-docs. Five members have more than 500 citations each, Profs. Tautgirdas Ruzgas, Gunilla Nordin Fredrikson, Thomas Arnebrant, Ann Wennerberg and Dr Sergey Schleev. Center members have also refereed papers for international journals on a regular basis. 6

7 Members of the Center take part in The National Research School of Odontology (Ann Wennerberg (coordinator), Gunnel Svensäter (local coordinator) and Julia Davies, supervisors), and The Research School in Pharmaceutical Sciences, LU (Johan Engblom, supervisor). Most researchers participate in undergraduate (the BMA and TELMah programmes) and/or graduate teaching (the BMMT, MS and Dentistry programmes), as well as PhD-student supervision. We strive to further integrate education (BSc, MSc, PhD), a cornerstone for the future of the Center. Particularly, one goal is to further integrate Master-level education into our research activities also at an operational level in specific projects. Research collaboration with industry is active in all four focus areas of the Center and during 2010 we attracted Novosense AB, Biogaia AB, ACO Hud Nordic AB, YKI AB and Eviderm Institute AB as partners in new research projects. Although border lines between the focus areas are not always clear cut, the company involvement distribute according to i) Eucaryotic cell-surface interactions (Promimic, PHI), ii) Molecular transport phenomena (AkzoNobel, YKI, ACO Hud Nordic, Eviderm Institute), iii) Molecular interactions at biointerfaces (Camurus, EuroDiagnostica, Promimic, Novosense, Anordica, Arcam, Bioglan, Galenica) and iv) Microbial biofilms (ArlaFoods, AnoxKaldnes, Gambro, Arcam, Biogaia). We also have close collaboration with Medeon AB and Medicon Valley Alliance. Center members are partners in the collaborative EU FP7 funded project Threedimensional nanobiostructure-based self-contained devices for biomedical application (Dr Sergey Shleev, coordinator) and continued to be involved in the EU FP6 Marie Curie Research Training Networks (MCRTNs), Bio-interfaces: from molecular understanding to applications. We are also involved in three EU Interreg programmes; Valorisation of knowledge intensive ideas in the South Baltic area (SB-VALOR), Öresund forum for innovation within nano-, bio- and medical technology (FinNBMT) and Öresund materials innovation comunity (Ö-MIC). Center members have been responsible for arranging three workshops, Biomaterials from fundamentals to Market Application (Biofilms 6 th Annual workshop), Biofilms Members Day and together with BIOSUM, Gothenburg; Biomaterials in medicine, (within the National Research School of Odontology), one salivary symposium at IADR in Barcelona and a kick off meeting for a new research project funded by KKs. The workshop Choice of and collaboration with CRO s in pharmaceutical development (launched by the Swedish Academy of Pharmaceutical Sciences) was held at Malmö University and co-organised by the Center. Center members have been active presenting their results on numerous occasions at national and international conferences and workshops. Center activities have also been visible through e.g. articles in PS Public Service Review European Union 19, a press release highlighting the 6 th annual workshop, and Malmö University newsletters. 7

8 3 List of Research Activities The research activities of the Center during 2010 may be described by the following headlines. Projects are listed with partners and funding in paranthesis. Projects in bold are funded by the center grant from KKs. In addition, we are also involved in three EU Interreg programmes; Valorisation of knowledge intensive ideas in the South Baltic area (SB-VALOR), Öresund forum for innovation within nano-, bio- and medical technology (FinNBMT) and Öresund materials innovation community (Ö-MIC). 3.1 Eucaryotic cell-surface interactions 1. Biomaterial 1(2), The influence of biochemical coat for implant bone incorporation, The in vivo part (Promimic AB, funded by KKs-Biofilms) 2. Cell-to-bio-mimetic interface interactions (funded by EU Marie Curie Research Training Networks) 3. Digital holography for cell studies (Phase Holographic Imaging AB, funded by the Crafoord foundation, the Magnus Bergwall foundation and Mah) 4. Biological responses to photo-reactive hydrophilic nano-size structures (funded by VR) 5. Hydrophilic and hydrophobic implant surfaces (funded by Vilhelm and Martina Lundgren Foundation) 6. Histological and 3-dimensional analysis of laminin coated polished ceramic implants (funded by Hjalmar Svensson Research Foundation) 7. The use of CaO as luting material and bone substitute (funded by VR) 8. Facilitation of soft tissue healing upon implant treatment in patients with supressed healing ability (funded by Mah) 3.2 Molecular transport phenomena 9. Adjuvants for products used in agriculture (AkzoNobel Surface Chemistry AB, funded by KKs-Biofilms) 10. Water a crucial factor in regulating biomembrane permeability (Physical Chemistry 1, LU, funded by FLÄK, LU) 11. Humectants and their mechanisms in skin (YKI AB, ACO Hud Nordic AB, Eviderm Institute AB, funded by KKs) 12. Miniature biofuel cells for self-contained bio-devices: electron transfer in threedimensional nanobiostructures (funded by VR & EU) 13. Grinfeld surface instabilities (funded by VR) 14. Effect of a gradient in water chemical potential on buccal drug delivery (Food technology, LU, funded by Mah) 3.3 Molecular interactions at biointerfaces 15. Mucoadhesion: Drug carrier interactions at biologically relevant interfaces (Camurus AB, funded by KKs-Biofilms) 16. Bioassay: New concept for lipid-based surface coatings in bioassays (EuroDiagnostica AB, funded by KKs-Biofilms) 8

9 17. Biomaterial 2(2), The influence of biochemical coat for implant bone incorporation, The in vitro part (Promimic AB, funded by KKs-Biofilms) 18. Development of biofuel cells for powering wireless transmission (Novosense AB, funded by KKs-Biofilms) 19. Attachable Diagnostic Devices with Individualised Referencing (ADDIR) (Galenica AB, funded by KKs-biofilms) 20. Symptomatic vs. asymptomatic atherosclerotic plaques (CRC, LU, funded in part by Mah) 21. Biocompatibility of metals (Anordica AB & Arcam AB, funded by KKs) 22. Hydration of mucous gel (funded by Mah) 23. Topical hydrogen peroxide in wound healing (Bioglan AB, pilot project) 24. Membrane impedance spectroscopy as a tool to study skin barrier function and variability (Galenica AB & Dermatology (LU), pilot project) 25. Development of novel multi-functional salivary substitutes for dry mouth syndrome patients (funded by the Swedish Laryng Foundation) 26. Screening of phase behavior in the DOPS/DOPE/water system and effects on lipid morphology from a decrease in water chemical potential (funded by CLRF) 3.4 Microbial biofilms 27. Milk Protein: Investigation of interactions between osteopontin and oral biofilm bacteria (Arla Foods AB, funded by KKs-Biofilms) 28. Carrier: Investigation and modelling of convection in biofilms for different carriers (AnoxKaldnes AB, funded KKs-Biofilms) 29. Probiotics: Effects of probiotic lactobacilli on biofilm formation and acid tolerance (Biogaia AB, funded by KKs-Biofilms) 30. Catheters: Biofilm formation on Peritoneal Dialysis catheters (Gambro Lundia AB, funded by KKs-Biofilms) 31. Biologically induced stress corrosion crack growth (Arcam AB, funded by KKs-Biofilms) 32. Biofilm activity as a marker to identify patients at risk of caries mechanisms underlying microbial stress tolerance (funded by KKs-Biofilms) 33. Caries prevention with fluoridated milk a prospective clinical and microbiological study of root caries (funded by Swedish Patent Revenue Foundation) 34. Biofilms on oral mucosal surfaces (funded by Swedish Dental Society) 35. Mucins and microbial biofilms a symbiotic relationship for health (funded by Mah and Crafoord Foundation) 36. Mucosal interactions as inducers of acid tolerance in oral microorganisms (funded by Crafoord Foundation and Swedish Patent Revenue Foundation) 37. Activities of microbial biofilms on bioactive implant surfaces (funded by Mah) 38. The plasminogen activating system interaction with microorganisms and a potential risk marker (funded by Swedish Dental Society) 9

10 4 Research highlights 4.1 Friction force spectroscopy for the study of the strength of protein layers Protein layers can confer diverse properties on surfaces such as molecule-binding, biocompatibility, or simply act as protective barriers against the surrounding medium. Considering the vast number of applications where they are involved, these layers can be subjected to multiple damage sources, including those of mechanical origin. Thus, the study of the mechanical properties of protein layers can provide with useful information for i) a better understanding of how the proteins interact both between themselves and with the underlying substrate, but also for ii) their design so that their resistance against mechanical damage is increased. During the last decade, the Atomic Force Microscope (AFM), where a sharp nm-sized tip is used to probe surfaces, has emerged as a powerful tool to study mechanical properties of protein layers. We have approached the study of these properties by a new methodology based on the Friction Force Spectroscopy (FFS) operation mode of the AFM, which allows studying the sample response to both normal and shear applied stresses. This methodology is based on the continuous two-dimensional scanning of a surface while ramping the applied load force. For each load force, a topography image of the scanned sample area and the average applied friction force during the scan are recorded. Therefore, FFS allows the characterization of the layers with friction-load curves, and with the characteristic topographies (like the initial rupture or the total removal) that correspond to the different regimes of the scratching process. This technique has been tested on β- and κ-casein monolayers. Caseins do not only act as natural emulsifiers in milk, but are also used as emulsifiers or dispersants in many technological and industrial applications. Our experiments showed that they can support pressures up to hundred of MPa before being removed while still exhibiting a high frictional behaviour, supporting their good performance as emulsifiers. Moreover, the technique was also proved to be able to study the dependence of the cohesion of the layers with properties of the surrounding liquid medium such as ph and ionic strength. Recent experiments on systems such as serum-proteins and saliva layers support the wide applicability of the technique. Figure 4.1. a) Schematic draw of a FFS measurement. b) and c) Topography images and frictionload curve corresponding to the scratching of a β- -casein monolayer. Sotres J, Svensson O and Arnebrant T., Friction force spectroscopy of β- and κ-casein monolayers. Langmuir 2011, 27(3),

11 4.2 Bioelectrocatalytic interfaces: Redox Enzymes Electronically Connected at Three-Dimensional Materials for Extracting Electrical Energy from Biofuels Bioelectrocatalytic devices, such as biosensors, have proven to be useful in different areas of applications including biomedicine. Current research in bioelectrochemistry is massively focused on the improvement of biofuel cells. The last year we have aimed to design three-dimensional (3D) conducting materials which, when loaded with redox enzymes, can constitute high power biofuel cells. Conducting 3D materials were based on redox hydrogels [1], carbon [2] or gold [3] nanoparticles, and nanostructured silica [4]. It was found that all these materials provide a possibility to control the procedures of assembly of 3D electrodes. The work was devoted to tune or adjust selectivity and optimize catalytic activity of the redox enzymes incorporated into the 3D structures of these electrodes. Recently developed 3D electrodes of biofuel cells show at least 10 times higher current densities if compare with 2D electrodes. 3D cathodes developed in our laboratory provide µa/cm 2 current densities. This means that a few micrometer thick biofuel cell of approximately 1 cm 2 area already now might power wireless biomedical devices. Figure 4.2. An example of 3D electrode based on micro-/ nanostructured silica layer. Relevant publications: 1. Design of a bioelectrocatalytic electrode interface for oxygen reduction in biofuel cells based on a specifically adapted Oscomplex containing redox polymer with entrapped Trametes hirsuta laccase. Ackermann, Y.; Guschin, D.A.; Eckhard, K.; Shleev, S.; Schuhmann, W. Electrochemistry Communications, 2010, 12(5), Stable floating air diffusion biocathode based on direct electron transfer reactions between carbon particles and high redox potential laccase. Shleev S.; Shumakovich G.; Morozova O.; Yaropolov A. Fuel Cells, 2010, 10(4), Laccase-gold nanoparticle assisted bioelectrocatalytic reduction of oxygen. Dagys, Marius; Haberska, Karolina; Shleev, Sergey; Arnebrant, Thomas; Kulys, Juozas; Ruzgas, Tautgirdas. Electrochemistry Communications, 2010, 12(7), Bioelectrochemical studies of azurin and laccase confined in three-dimensional chips based on gold-modified nano- /microstructured silicon. Ressine A., Vaz-Dominguez C., Fernandez V.M., De Lacey A.L., Laurell T., Ruzgas T., Shleev S. Biosensors and Bioelectronics, 2010, 25(5), The following projects support the development of 3D biofuel cells: 1. Wireless self-powered biodevices: Function of nanowired multicentre redox enzymes and living cells", Main applicant: Sergey Shleev. The Swedish Research Council, project number: Bioelectrochemical 3D nanobiostructures in physiological liquids and cell based in-vitro platforms Main applicant: Tautgirdas Ruzgas. The Swedish Research Council, project number: Three-dimensional nanobiostructure-based self-contained devices for biomedical application, Coordinator: Sergey Shleev. EU FP7-NMP-2008-SMALL-2. Grant Agreement Number:

12 4.3 Factors affecting transport of tebuconazole over leaf cuticle The complex structure of plant cuticles constitutes the main barrier for fungicide uptake in leaves. Consequently, the detailed underlying mechanism of action of adjuvants, often used to facilitate fungicide permeation in leaves, is also complex and remains to be resolved. The overall aim of this project is thus to obtain a better understanding of the mechanisms of action of specific surfactants, in order to facilitate the design of better adjuvants for agricultural fungicides. We have combined in vitro diffusion methodology (Franz cells) with sorption isotherms and membrane impedance spectroscopy to evaluate the effect of two specific adjuvants (C 10 EO 7 and C 8 G 1.6 ) on the bioavailability of tebuconazole as model active ingredient, using the adaxial side of leafs from the model plant Clivia Miniata Regel as the membrane. The barrier properties of plant leaves may respond to external factors, like changes in ambient relative humidity or gradients imposed by individual formulations applied and therefore, inert silicone membranes were employed in parallel to plant membranes to distinguish between factors affecting the diffusion coefficient in the membrane (D i ) and the gradient in chemical potential (d i /dz) over the membrane. From the data given below it is evident that fungicide permeation over silicone membrane is strictly dependent on the gradient in tebuconazole chemical potential (Figure 4.3.1), while the prescence of surfactants has a strong impact on the diffusion coefficient of tebuconazole in Clivia cuticle, resulting in up to four times higher permeability (Figure 4.3.2). C 10 EO 7 is more effective than C 8 G 1.6 in promoting tebuconazole permeation through the Clivia cuticle, and C 10 EO 7 also has a more pronounced ability to decrease the extremely long lag-time. Moreover, the Clivia cuticle can accommodate large amounts of tebuconazole and thereby act as a depot over time. We have also shown using impedance spectroscopy that the barrier properties of Clivia cuticle improves with maturation of the leaf. Figure Tebuconazole permeability over a silicone membrane. Carrier: water, and watersurfactant (4%) mixtures. a teb = Figure Tebuconazole permeability over a Clivia membrane. Carrier: water, and watersurfactant (4%) mixtures. a teb = Factors Affecting Transport of Tebuconazole over Silicone Membrane and Leaf Cuticle, Fagerström A, Kocherbitov V, Lamberg P, Bergström K, Westbye P, Ruzgas T and Engblom J. in 9th International Symposium on Adjuvants for Agrochemicals, ISAA Society; Baur P and Bonnet M Eds. August 2010, Pages ISBN Effects of adjuvants on Tebuconazole leaf cuticle penetration, Fagerström et al, Manuscript to be submitted Surfactant induced fluidization of plant leaf cuticle, Fagerström et al, Manuscript to be submitted

13 4.4 Low levels of fluoride inhibit acid tolerance of plaque bacteria in vivo Fluoride is used for prevention of dental caries mainly due to its potential to decrease solubility and promote remineralisation of enamel and root dentin. In vitro studies show that fluoride also affects the physiology of oral streptococci. However, data on the effect of fluoride on acid tolerance in plaque in vivo is limited. The aim of this investigation was to study the effect of fluoride on plaque acid tolerance and composition, lactic acid production, and the clinical effect on re-mineralization of root caries lesions. The test group (F-group) consumed 200 ml of cow s milk supplemented with 5 mg/l NaF as a single dose once per day, the milk control group (Mgroup) drank 200 ml of unsupplemented cow s milk and a no-milk control group (C-group) did not consume milk in this manner. The length of the study was 15 months. Dental plaque samples were taken at baseline and after 15 months. The proportion of acid tolerant bacteria in plaque was estimated using LIVE/DEAD BacLight TM staining after exposure to ph 3.5 for 2 hours. Lactic acid production after glucose pulsing was measured enzymatically. The Electronic Caries Monitor (ECM) was used to measure the electrical resistance of root surface lesions. Plaque from subjects in the F-group showed a statistically significant decrease in plaque acid tolerance and a significant increase in ECM values were found after 15 months compared to baseline indicating re-mineralization. Lactic acid production from glucose was also lower in the F-group although not statistically significant. This experimental clinical study shows that daily intake of fluoride reduces plaque acid tolerance and lactic acid production in vivo and promote remineralization of root caries lesions. BASELINE AFTER 15 MONTHS DECREASED PLAQUE ACID TOLERANCE INCREASED PLAQUE ACID TOLERANCE Figure 4.5. Bacteria in plaque stained with LIVE/DEAD BacLight TM staining. Green cells are acid tolerant while red cells are not. Neilands J., L.G. Petersson, D. Beighton and G. Svensäter. Fluoride inhibits acid tolerance of root surface biofilms. Manuscript 13

14 4.5 Peritoneal dialysis catheters show presence of bacteria without clinical signs of infection Fifteen peritoneal dialysis catheters extracted from patients undergoing renal transplantation (i.e. from patients with no clinical signs of infection) were investigated for the presence of bacteria using microbiological culture and confocal laser scanning microscopy (CLSM). For reference, two catheters from patients with infections were also investigated. The results showed 82% of the catheters to be colonised by bacteria, although in a rather low numbers. The bacteria were heterogeneously spread all over the catheter surface and several species often colonised the same area (Figure 1). The major species found were Staphylococus epidermidis and Propionibacterium acnes, but several others were also detected, including Micrococcus spp, Staphylococcus lugdunensis, Staphylococcus warneri, Corynebacterium spp, Proteus mirabilis, Rothia mucilaginosa, Streptococcus sanguis and Staphylococus aureus. Figure 4.6. Species found on peritoneal dialysis catheters positive in the microbiological cultures. Pihl M, Davies JR, Johansson A-C and Svensäter G. Occurrence of bacteria on catheters in patients undergoing peritoneal dialysis Submitted to Journal of Medical Microbiology. 14

15 4.6 Adhesion of Streptococcus oralis to titanium surfaces - effects of surface roughness and conditioning films Initial healing is a critical phase in dental implant therapy and optimum surface roughness is one of the key factors of importance for successful osseo-integration. Surfaces designed to promote osteoblast activity may however also enhance bacterial adhesion and stimulate microbial activity thus increasing the risk of peri-implant infections in the longer-term. The aim of this investigation was to determine how surface roughness affects the adhesion of Streptococcus oralis (a species frequently isolated from peri-implant infections) to titanium surfaces used in dental implants in the presence of conditioning films derived from saliva and serum. Titanium plates with average surface roughness (Sa) of 0,5 or 1,5 µm, uncoated or coated with 25% whole saliva or 5% human serum were exposed to exponential growth phase cells of Streptococcus oralis (LA 11) in a flow-cell system for 2 h. After washing for 1h, the numbers of adhered bacteria were assessed using confocal laser scanning microscopy (CLSM) after staining with the Baclight Live/Dead kit (Figure 1). The mean percentage bacterial coverage on the uncoated smooth surface (Sa = 0.5 µm) was 2±1% while that on the moderately rough surface (Sa = 1.5 µm) was significantly greater (mean percentage coverage = ± 0.88%, p < 0.01). Figure 4.7. CLSM images showing biofilms formed on moderately rough titanium surfaces in (a) the absence of a conditioning film or in the presence of (b) a saliva- or (c) and serum-derived conditioning film. For both the moderately rough and smooth surfaces, a conditioning film of saliva significantly increased the adhesion of bacteria (p< 0.01) whereas bacterial binding in the presence of a serumderived film showed no differences to that on the control (uncoated) surface. These data suggest that increased surface roughness, as well as the presence of salivary proteins, are important determinants of the level of colonization by streptococci on oral implant surfaces. Dorkhan M, Chávez de Paz M, Svensäter G and Davies JR. Adhesion of Streptococcus oralis to turned and blasted titanium surfaces - effects of saliva- and serum-derived pellicles. Manuscript 15

16 4.7 How to detect protease activity of dental biofilms in situ Proteolytic bacteria export proteases to their immediate surroundings. The extracellular proteolytic activity of these bacteria can thus be monitored through the addition of a protease substrate to the culture medium. Casein is a substrate for the four major types of proteases (serine, aspartic, cysteine and metalloproteases) and is thus a suitable substrate for screening protease activity. Using fluorescein-labeled casein, protease activity can be detected. Proteolytic activity of bacteria grown in biofilms has been visualized by confocal microscopy after addition of fluorescein-labeled casein. Figure 4.8. Proteolytic activity of bacteria in a clinical subgingival biofilm sample visualized as green fluorescence. Kinnby B, Wickström C & Svensäter G, Method development

17 4.8 Selective adhesion and phenotypic changes in oral streptococci revealed through interaction with mucinconditioned surfaces The resident microflora of dental plaque changes as it matures over time. Different streptococcal species are suggested to adhere and colonize at different time points in the maturation process, where early colonizers have the ability to adhere and grow, using the host proteins as substrates and late colonizers require other bacteria or their products to be able to adhere and grow. We show here, that two streptococcal species, Streptococcus mitis biovar 2 and Streptococcus mutans, display very different behaviors when introduced to surfaces conditioned with human salivary mucin MUC5B, the major glycoprotein found in the mucus film covering all oral surfaces. Using test surfaces conditioned with the salivary MUC5B mucin, S. mitis biovar 2 showed avid adherence as well as a phenotypic shift towards more protease active cells. When introduced to surfaces conditioned with other salivary proteins, this effect was not seen, suggesting a specific interaction between S. mitis biovar 2 and the MUC5B molecule. However, when S. mutans was studied under the same conditions, very few cells adhered to the conditioned surfaces, MUC5B or the other salivary proteins, suggesting an inability of S. mutans to adhere to the natural occurring salivary film components found in vivo. The same increase in protease active cells observed for S. mitis biovar 2 was found in the S. mutans cells, although there was no difference between the two substrates. This work clearly shows a specific difference between two streptococcal species in the way they adapt to different environments, S. mitis biovar 2 being able to adhere and colonize a salivary conditioning film, whereas S. mutans cannot. Figure 4.9. Adhesion of S. mitis biovar 2 (A) and S. mutans UA159 (B) to surfaces coated with salivary proteins, MUC5B or low-density salivary proteins. Bacteria were allowed to adhere to coated or uncoated surfaces for 2 hours and attached cells were visualized in a CLSM after BacLight LIVE/DEAD staining. * Differences are statistically significant (p < 0,05) using the Mann-Whitney t-test. Christian Kindblom, Gunnel Svensäter & Claes Wickström Salivary proteins influence phenotypic changes in Streptococcus mutans and Streptococcus mitis biovar 2 biofilm cells differences in adhesion and protease activity. To be submitted. 17

18 4.9 Spatial distribution of multiple species in complex biofilm communities In nature, bacterial biofilm communities are highly organized structures composed of multiple species that are believed to interact with each other in order to coexist. Synergistic coexistence of bacteria in mixed biofilm communities is suggested to play a crucial role in development of chronic infections as many species can become extremely resistant against antimicrobials and host defences. To better understand the spatial structure of multiple species communities it is necessary to visually characterize the distribution of individual species. We have developed an automated in vitro system to study spatial distribution of bacteria in multi-species biofilm communities. Our system is based in a combination of fluorescence in situ hybridization (FISH), confocal laser scanning microscopy (CLSM), and digital image analysis. FISH probes targeting the 16S rrna gene are designed to verify the abundance and spatial location of microbial community members. Overall, the knowledge gained by this method about distribution and interactions in multi-species biofilms will facilitate the rapid analysis of microbial communities in the sense of assessing changes in microbial populations as a function of time or environmental conditions. Prospectively, this methodology can be applied in combination with specific fluorescent markers targeting metabolic processes to allow the investigation of in situ structure/function analysis of complex microbial communities. A B Figure Spatial distribution of two oral biofilm communities. (A) A four-species biofilm community composed of clinical root canal isolates of Lactobacillus salivarius (red), Streptococcus gordonii (green/yellow), Actinomyces naeslundii (blue) and Enterococcus faecalis (violet). (B) A three-species biofilm community composed of supra-gingival isolates of Lactobacillus fermentum (red), Streptococcus gordonii (green/yellow) and Actinomyces naeslundii (blue). Bar = 10 µm. Chavez de Paz L,

19 4.10 On Ca2+ incorporation and nanoporosity of titanium surfaces and the effect on implant performance Implants need to perform in three biological arenas: in relation to bone-tissue, soft-tissue, and microbial biofilms. An implant should be properly osseointegrated and have a tight adaptation of surrounding soft-tissues but it should at the same time not be prone for extensive biofilm formation or be difficult to clean. More specifically we aimed for: Bone: To evaluate the importance of anodic oxidation and Ca2+ incorporation/surface chemistry of commercially pure titanium implants regarding osseointegration, and whereas the chemical modification may compensate for a minimal surface roughness. Oral mucosa: To evaluate the effect of sol-gel derived nanoporous TiO2 coating of commercially pure titanium for the adaptation of oral mucosa. Bacterial adhesion and biofilm formation: To investigate bacterial adhesion, as well as biofilm formation and retention of oral bacteria in vitro on smooth and moderately rough, anodized and Ca2+ incorporated, as well as, nanoporous surfaces. Figure Bone adhesion towards Ca ion implanted surfaces, in trabecular and cortical bone Figure Soft tissue adhesion demonstrated with TEM We have shown that surface chemistry/anodic oxidation and Ca2+ incorporation of titanium surfaces may enhance the osseointegration and could possibly compensate for a minimal surface roughness. Nanoporous TiO 2 coating indicates some advantages in relation to unmodified titanium regarding the sealing of oral mucosa. A tendency of increased biofilm accumulation of oral bacteria in vitro was found for moderately rough (Sa 1-2 μm) blasted surfaces compared to smooth ones (Sa <0.5 μm). Moderately rough surfaces, in addition, retained more bacteria after mechanical removal of adhered biofilms compared to smooth. Nanoporosity or Ca2+ incorporation did not affect the bacterial adhesion or biofilm formation compared to turned surfaces. Increased bone contact to a Ca2+ incorporated oxidized c.p. titanium implant: an in vivo study in rabbit. Fröjd V, Franke-Stenport V, Meirelles L, Wennerberg A. Int J Oral Maxillofac Surg 37:6 (2010) Importance of Ca2+ modifications for osseointegration of smooth and moderately rough anodized titanium implants a removal torque and histological evaluation in rabbit. Fröjd V, Wennerberg A, Franke-Stenport V. Clin Impl Dent Rel Res Nanoporous TiO2 thin film on titanium oral implants for enhanced human soft tissue adhesion - a histological evaluation in three different levels of resolution. In situ analysis of biofilm formation on titanium surfaces Fröjd V, Chávez de Paz L, Andersson M, Wennerberg A, Davies J, Svensäter G. Submitted. Microbial biofilm formation on smooth nanoporous TiO2 coated and anodized Ca2+ modified surfaces. Fröjd V, Linderbäck P, Wennerberg A, Chávez de Paz L, Svensäter G, Davies J. Submitted. 19

20 4.11 Biologically induced stress corrosion as an indicator of residual stress. In general, surface instabilities play an important role in industry. The applications are initial stages of biologically induced corrosion. The examples from nuclear, petroleum and offshore industry are numerous. Problems arise during manufacturing and in operation because of high stresses and exposure to aggressive environment. As much as 25% of all accidents reported to the Swedish Plant Inspectorate are claimed caused by stress corrosion. A large part of these can be attributed to biological induced corrosion. A severe circumstance is that the cracks propagate at very small loads. Occasionally the general status of the environment is sufficient to cause stress corrosion, but in general the extreme local micro-environment under microbial colonies are responsible for so called pitting and initiation of edge cracks that subsequently grow into the structure. Apart from the immediate advantage of more knowledge, several applications taking advantage of the possibilities for non destructive testing of stresses could have been identified. The most striking example is that inspection of a developing surface roughness has been proven to work as a tool to discover high mechanical stresses and risk zones for corrosion cracks. Theoretical studies of an amorphous material explain the mechanism behind the repeated branching of cracks that is observed for cracks in aggressive environment. Numerically simulated crack growth was performed using a moving boundary finite element formulation. The results show great agreement with the experiments. An observed scatter in results in both experiments and in the numerical simulations reflects an inherent perturbation sensitivity of environmentally assisted cracking. Figure Left) Typical initiation pattern of small pits resulting from an aggressive environment. The pit density of pits reveal the mechanical stress in the structure. Right) Maximal principal strain field surrounding a newly branched crack. The arrows indicate where the surface straining equals the threshold strain. On initiation of chemically assisted crack growth and crack propagation paths of branching cracks in polycarbonate, Hejman, Ulf, 2010, Licentiate Thesis, 77 pages, Media-Tryck AB, Lund Sweden Dissolution driven crack branching in polycarbonate, Hejman, Ulf, Bjerkén, Christina, Fatigue and Fracture of Engineering Materials and Structures, 2010 Environmentally assisted initiation and growth of multiple surface cracks, Hejman, Ulf, Bjerkén, Christina, International Journal of Solids and Structures, 14-15, vol. 47, p ,