BIO FILMS INTERFACES. Biofilms Research Center for Biointerfaces

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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 #5 January 1 st, 2009 December 31 st, 2009

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 in the associated liquid phase. Contact information Biofilms Research Centre 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: University Hospital MAS (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 Håkan Ericsson, Assoc. Prof., Vice Director 2009 Thomas Arnebrant, Prof., Director 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. Vitaly Kocherbitov, Assoc. Prof. Liselott Lindh, Assoc. Prof. Anette Gjörloff-Wingren, Assoc. Prof. Zoltan Blum, Assoc. Prof. Lars Olsson, Assoc. Prof. Liu-Ying Wei, Assoc. Prof. Julia Davies, Assoc. Prof. Bertil Kinnby, Assoc. Prof. Tove Sandberg, Dr. Maria Stollenwerk, Dr. Christina Bjerkén, Dr. Sergey Shleev, Dr. 1.2 Junior researchers and postdocs Olof Svensson, Dr. Olga Santos, Dr. Tobias Halthur, Dr. Anna Ketelsen, Dr. Laura Varas, Dr. Claes Wickström, Dr. Jessica Neilands, Dr. Luis Chavez de Paz, Dr. Javier Sotres, Dr Ida Svendsen 1.3 PhD students Jildiz Hamit Eminovski Linda Andersson Peter Hellman Ulf Hejman Alma Masic Maria Pihl Sebastian Björklund (LU) Anton Fagerström Yana Znamenskaya 1

4 Adnan Safdar Christian Alfredsson Kindblom Marjan Dorkhan Kostas Bougas Magnus Falk 1.4 Technical and administrative staff Eva Nilsson, Administrative coordinator Ulrika Troedsson, Technician Agnethe Henriksson, Technician Madeleine Blomqvist, Technician 1.5 MSc students 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 & CTS Maihemutijiang Maimaiti Carl Mikaelsson Ajigul Nuermaimaiti Oyetunji Oladele Kazeem Wureguli Reheman Christian Ukoha Oji Erik Öberg Simayijiang Zhayida MS Master at HS & CTS Adili Maimaiti Guzainuer Maimaitiyiming Huaizhi He Maimaitiyili Tuerdi Hao Qin BMMT Master at HS Noor Al-Attar Fozia Elahi Tannaz Horrieh Charlotte Lerbech Jensen Shamila Khan 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. 2

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 Pro-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 Håkan Eriksson, Assoc. Prof., Vice Director 2009 Per Ståhle von Schwerin, Prof. Gunnel Svensäter, Prof. Ann Wennerberg, Prof. Gunilla Nordin-Fredrikson, Prof. Steering group Eva Engquist, Pro Vice-Chancellor, Malmö University, Chairman Margareta Östman, Prof., Dean Faculty of Health and Society Naser Eftekharian, Head School of Technology Malmö University Lars Matsson, Prof., Dean Faculty of Odontology (- June 2009) Lars Bondemark, Prof., Dean Faculty of Odontology (July ) 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 Krister Thuresson, Dr., Director CMC Regulatory Affairs Camurus AB (- October 2009) Markus Johnsson, Dr., Senior Director Pharm. Development Camurus AB (November ) Eva Engquist, Pro.Vice-Chancellor, Malmö University Zoltan Blum, Assoc. Prof., Malmö University 3

6 2 The Director s Report During 2009 the Center project portfolio has conatained 34 projects, of which 13 are funded through our center grant from KKs. A major emphasis in 2009, apart from conducting high quality front line research, has been to secure additional funding. The efforts have been fruitful with a 94% increase in external funding compared to Some of the new projects bring new industry partners into the Center, and some also facilitate our ambition to increase focus on medical applications and to improve involvement of preclinical and clinical sciences. Linda Andersson, Ida Svendsen and Peter Hellman successfully defended their PhDtheses, as did Karolina Haberska and Marie Friberg (enrolled at LU, with supervisors at Mah). Jildiz Hamit Eminovski successfully defended her licentiate thesis. Ida Svendsen got the award for the best PhD-thesis at Malmö University Her thesis In vitro and in vivo studies of salivary films at solid/liquid interfaces is a good example of the potential of interfaculty collaboration within Biofilms-Research Center for Biointerfaces. Associate Professor Birgitta Rosén (Handsurgery, LU) has been part time employed as guest teacher until March Professor Anders Heyden and Drs Niels-Christian Overgaard and Tobias Halthur left their positions during the spring. Drs Javier Sortres and Olga Santos where employed in September. 10 new PhD-students where enrolled during the year, seven at Malmö University and three at Lund University, with supervision from Malmö. During 2009 we have produced 51 publications in international journals and 6 proceeding papers. Publications are in journals such as Trends in Immunology (9.910), Journal of Controlled Release (5.690), The International Journal of Cancer (4.734), Materials Science and Engineering (2.201) and FEMS Microbiology and Immunology (1.972). The impact factors span from to indicating a substantial scientific quality of the research carried out within the Center. In addition to this approximately 21 oral and 25 poster presentations were made at national and international meetings. In reviewing the number citations over the past six years on papers produced by the Center members we sum up 6800 for permanent staff and 900 for junior researchers and post-docs. Four members have more than 500 citations each, Profs. Tautgirdas Ruzgas, Gunilla Nordin Fredrikson, Thomas Arnebrant and Dr Sergey Schleev. Center members have also refereed more than 60 papers for international journals. Members of the Center take part in The National Research School of Odontology (Prof. Gunnel Svensäter (coordinator), Prof Ann Wennerberg and Assoc. Prof Julia Davies, supervisors). 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. 4

7 Research collaboration with industry is active in all four focus areas of the Center and during 2009 we attracted Promimic AB, Bioglan AB and Nares AB as new partners. 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) Transport phenomena (AstraZeneca, AkzoNobel, Camurus, Galenica), iii) Molecular interactions at biointerfaces (Camurus, SinclairPharma, EuroDiagnostica, Promimic, TetraPak, Stora Enso, Anordica, Arcam, Bioglan, Nares) and iv) Microbial biofilms (ArlaFoods, AnoxKaldnes, SinclairPharma, Gambro, Arcam). We also have close collaboration with Medeon AB and Medicon Valley. Center members involve in the collaborative EU FP7 funded project Three-dimensional nanobiostructure-based self-contained devices for biomedical application (Dr Sergey Shleev, coordinator) and continue to be involved in the EU FP6 Marie Curie Research Training Networks (MCRTNs), Bio-interfaces: from molecular understanding to applications. We also involve in the EU South Baltic program VALOR Valorisation of knowledge intensive ideas in the South Baltic area. A new QCM-D was purchased during the year, a valuable complement for our activities regarding thin films formation. Through a generous donation from AstraZeneca we received an X-ray diffraction camera (Kratky SWAXD) and two additional HPLC-UV set-ups, most useful in both research and education activities Center members have been responsible for arranging two workshops, Microarrays (Biofilms 5 th Annual workshop) and Oral Biofilm and Risk (National Research School of Odontology), and one PhD-student day with invited mentors. The students now also meet on private initiatives, which is very encouriaging from a cross faculty collaboration perspective. Furthermore, kick-off meetings were arranged in Malmö within the two new EU-projects. Together with Medicon Valley Alliance and Trial Form Support AB we arranged a meeting to present a new education program to industry partners. 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 two press releases, Rapidus Newsletter and Malmö University newsletters. 5

8 3 List of Research Activities The research activities of the Center during the period of report 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 also involve in the EU South Baltic program VALOR Valorisation of knowledge intensive ideas in the South Baltic area. 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) 3.2 Molecular transport phenomena 4. Hydration. Interactions between pharmaceutical materials and water (AstraZeneca, funded by KKs-Biofilms) 5. Adjuvants for products used in agriculture (AkzoNobel Surface Chemistry AB, funded by KKs-Biofilms) 6. Stress driven diffusion (pilot project funded by Mah ) 7. Water a crucial factor in regulating biomembrane permeability (Physical Chemistry 1, LU and Camurus AB, funded by FLÄK, LU) 8. Miniature biofuel cells for self-contained bio-devices: electron transfer in threedimensional nanobiostructures (funded by VR & EU) 9. Grinfeld surface instabilities (funded by VR) 10. Stress corrosion (funded by VR) 11. Set up and optimisation of an in vitro mucosa permeation model (Food technology, LU, pilot project) 3.3 Molecular interactions at biointerfaces 12. Mucoadhesion: Drug carrier interactions at biologically relevant interfaces (Camurus AB, funded by KKs-Biofilms) 13. Antiplaque 1(2): Adsorption and biofilm formation at oral interfaces (SinclairPharma AB, funded by KKs-Biofilms) 14. Bioassay 1(2): New concept for lipid-based surface coatings in bioassays (EuroDiagnostica AB, funded by KKs-Biofilms) 15. Bioassay 2(2): New concept for lipid-based surface coatings in bioassays (EuroDiagnostica AB, funded by KKs-Biofilms) 16. Biomaterial 2(2), The influence of biochemical coat for implant bone incorporation, The in vitro part (Promimic AB, funded by KKs-Biofilms) 6

9 17. Symptomatic vs. asymptomatic atherosclerotic plaques (CRC, LU, funded in part by Mah) 18. Fracture of biofibre-, polymer- and metal composites (TetraPak AB & Stora Enso AB, funded by KKs) 19. Biocompatibility of metals (Anordica AB & Arcam AB, funded by KKs) 20. Hydration of mucous gel (funded by Mah) 21. Topical microemulsion as nasal drug delivery system (Bioglan AB & Nares AB, pilot project) 3.4 Microbial biofilms 22. Milk Protein: Investigation of interactions between osteopontin and oral biofilm bacteria (Arla Foods AB, funded KKs-Biofilms) 23. Carrier: Investigation and modelling of convection in biofilms for different carriers (AnoxKaldnes AB, funded KKs-Biofilms) 24. Antiplaque 2(2): The effect of surface coatings on bacterial biofilm formation (SinclairPharma AB, funded KKs-Biofilms) 25. Biofilm formation on Peritoneal Dialysis catheters (Gambro Lundia AB, funded by KKs-Biofilms) 26. Biologically induced stress corrosion crack growth (Arcam AB, funded by KKs-Biofilms) 27. Caries prevention with fluoridated probiotic milk a prospective clinical and microbiological study of root caries (funded by Swedish Patent Revenue Foundation) 28. Survival strategies of bacteria in oral biofilms (funded by Swedish Research Council) 29. Mucins and microbial biofilms a symbiotic relationship for health (funded by Mah and Crafoord Foundation) 30. Mucosal interactions as inducers of acid tolerance in oral microorganisms (funded by Crafoord Foundation and Swedish Dental Society) 31. Biofilm activity as a marker to identify patients at risk of caries mechanisms underlying microbial stress tolerance (funded by Mah) 32. Activities of microbial biofilms on bioactive implant surfacrs (funded by Mah) 33. The plasminogen activating system interaction with microorganisms and a potential risk marker (funded by Crafoord foundation and Swedish Dental Society) 7

10 4 Research highlights 4.1 A water gradient can be used to regulate drug transport across skin At normal conditions there is a substantial water gradient over the skin as it separates the water-rich inside of the body from the dry outside. This leads to a variation in the degree of hydration from the inside to the outside of skin and changes in this gradient may affect the structure and function of skin. Sebastian Björklund et al have shown that a water gradient can be used to regulate transport of drugs with different lipophilic characteristics across the skin barrier. The transport of metronidazole (log P o/w =0.0) and methyl salicylate (log P o/w =2.5) across skin increases abruptly at low water gradients, corresponding to high degrees of skin hydration, and that this effect is reversible.this phenomenon is highly relevant to drug delivery applications. Further, the results contribute to the understanding of the occlusion effect and indicate the boundary conditions of the water gradient needed to make use of this effect. Björklund S, Engblom J, Thuresson K and Sparr E, A water gradient can be used to regulate drug transport across skin, J Control Release, in press Steady-state flux / µg cm -2 h -1 a w, d a w, d 0,8 0,85 0,9 0, PEG PEG 4000 Dextran Steady-state flux / µg cm -2 h ,8 0,85 0,9 0, PEG 1500 PEG 4000 Dextran µ w, d / J mol -1 µ w, d / J mol -1 Cumulative mass per area / µg cm µ 400 w, d -600 µ w, d -13 a w, d a w, d µ w, d -600 µ w, d -13 a w, d a w, d Time / h Cumulative mass per area / µg cm µ w, d -600 µ µ w, d -13 w, d -600 µ w, d -13 a 400 w, d a a w, d w, d a w, d Time / h Figure 4.1. Effect of a water gradient on metronidazole permeability in vitro over corresponding excised skin (left) and silicone membrane (right) 8

11 4.2 Adsorption and elution of salivary films from solid surfaces The salivary pellicle is formed by selective adsorption of salivary proteins on tooth enamel. It has lubricating and protective functions, which may be altered during plaque (biofilm) control treatments. In order to develop products aimed at controlling e.g. plaque growth or to develop new or improved salivary substitutes it is important to get a better understanding of protein behaviour at the interface. We have therefore investigated the interfacial properties of human salivary films formed on silica and hydroxyapatite (HA) surfaces in different adsorption media (PBS and water) by the use of quartz crystal microbalance with dissipation (QCM-D) and ellipsometry. We observed that saliva adsorbs at a higher extent onto the HA surfaces compared to the silica surfaces, regardless of the adsorption media. For both surfaces a higher adsorption of saliva was obtained when the adsorption media was PBS compared to water. Since Sodium dodecyl sulphate (SDS) is a common surfactant used in many types of oral care products we have also investigated how SDS solutions interact with the saliva film at different SDS concentrations. We observed that SDS was able to remove the saliva film completely from the silica surfaces but only partially from the HA surfaces. As expected, the highest pellicle removal from both surfaces was observed at the concentration corresponding to the cmc. Our results also implied that a softer saliva layer was build up in PBS for both surfaces. In water a softer saliva layer was formed onto the HA surfaces, while in PBS the saliva layer formed at both surfaces seemed to have similar structure. Olga Santos, Liselott Lindh, Tobias Halthur, and Thomas Arnebrant, Adsorption behaviour and elution of saliva from silica and hydroxyapatite surfaces with SDS and delmopinol studied by quartz crystal microbalance. Manuscript. SDS 0.005% 0.05% 0.5% SDS 0.005% 0.05% 0.5% 0 HWS Rinse Rinse Rinse Rinse 14 HWS Rinse Rinse Rinse Rinse 12 Silica Hydroxyapatite f 5 (Hz) PBS Silica D 5 (10-6 ) PBS 2-80 Hydroxyapatite Time (s) Time (s) Figure 4.2. Adsorption of saliva pellicle on silica and HA surfaces in PBS and elution with SDS 9

12 4.3 Bioelectrocatalytic interfaces: Redox Enzymes Electronically Connected at Three-Dimensional Materials for Extracting Electrical Energy from Biofuels Bioelectrochemical devices, such as biosensors, have proven to be useful in different areas of applications including biomedicine. Current research in bioelectrochemistry is massively focused on development of biofuel cells and defining principals of biocomputing devices. During the last year at our department we have focused on the studies of bioelectrocatalytic function of enzymes in three-dimensional (3D) conducting materials with the purpose to increase the power density of biofuel cells. Two major approaches were pursued, i.e. layer by layer deposition of bioelectrocatalytic enzyme nanoparticle conjugates [1] and making conducting 3D nanostructures from silicon [2]. Direct and mediated electron transfer reactions between different enzymes, viz. blue multicopper oxidases (laccase [2] and ceruloplasmin [3]) and cellobiose dehydrogenase [4] on planar and 3D electrodes were compared to understand the mechanism of functioning of both cathodic (oxygen reducing enzymes) and anodic (glucose oxidizing enzymes) bioelements. As a final outcome a biofuel cell operating in human serum (see figure below) was constructed. The cell exploits the principal of direct electron transfer for bioelectrocatalysis on both anode and cathode. As a consequence of this the biofuel cell is simple and does not contain leaking and toxic redox mediators [5]. 1. Haberska K. Protein and polyelectrolyte layer-by-layer films: Assembly and electron transfer. Doctoral dissertation, Lund University, Lund, 2009, pp Ressine A., Vaz-Dominguez C., Fernandez V.M., De Lacey A.L., Laurell T., Ruzgas T., Shleev S. Bioelectrochemical studies of azurin and laccase confined in three-dimensional chips based on gold-modified nano-/microstructured silicon. Biosensors and Bioelectronics, 2010, 25(5), Haberska K., Vaz-Domínguez C., De Lacey A.L., Dagys M., Reimann C.T., Shleev S. Direct electron transfer reactions between human ceruloplasmin and electrodes. Bioelectrochemistry, 2009, 76(1-2), Stoica L., Ruzgas T., Gorton L. Electrochemical evidence of self-substrate inhibition as functions regulation for cellobiose dehydrogenase from Phanerochaete chrysosporium. Bioelectrochemistry, 2009, 76(1-2), Coman V., Ludwig R., Harreither W., Haltrich D., Gorton L., Ruzgas T., Shleev S. A direct electron transfer-based glucose/oxygen biofuel cell operating in human serum. Fuel Cells, 2009, DOI: /fuce blue multicopper oxidase cellobiose dehydrogenase O H gluconolactone 2 H 2 O FAD 2 glucose T2/T3 I red I ox T1 I red I ox 4 e - Heme 4 e V 10

13 4.4 Hydration of proteins: water sorption isotherm of lysozyme Investigations the hydration of proteins is an important part of protein science. The hydration of proteins can be studied by two different approaches. In one approach, protein-water interactions are studied in dilute solutions of proteins in water. In the other approach, changes in structural, dynamic, and thermodynamic properties of proteins are monitored as functions of water content at relatively low hydration levels. Water sorption isotherms of proteins are usually interpreted with such models as BET or GAB that imply the formation of multilayers at solid-gas interface. However, this approach is not applicable to globular proteins such as humid lysozyme where a solid-gas interface does not exist. We proposed to interpret the water sorption isotherms of proteins considering adsorption of water at protein-protein interface. Thermodynamic analysis based on experimental data on enthalpy and entropy of hydration of lysozyme showed that the Langmuir sorption isotherm can be used to describe hydration of protein-protein interface. The result of the fitting of the sorption isotherm of lysozyme with the Langmuir model shows good agreement with the structural data on lysozyme. Hydration of lysozyme: the protein-protein interface and the enthalpy-entropy compensation. Langmuir, DOI: /la903210e Figure 4.4. Schematic structures of lysozyme in solution (left) and at a low hydration level (right). The structure of lysozyme in solution is from the protein database (193 L); water molecules are not shown. The low-hydration-level structure is for illustration purposes only and does not correspond to any experimentally obtained structure 11

14 4.5 Effect of Hydration on Structural Properties of Mucous Gel The mucus barrier and its transport properties are essential for proper functioning of the digestive, respiratory and reproductive systems of vertebrates, including humans. From an engineering point of view, mucus is an outstanding water-based lubricant. The principal components of mucus are the glycoprotein mucin and water. Mucin forms the macromolecular matrix of mucus and dominates its rheological properties. Mucin molecules obtained from different sources have different structures ranging from bottle brush to dumbbell type structures. It is known from literature that mucin can form liquid crystalline phases but exact phase behaviour of mucin at different temperatures and hydration levels has yet to be determined. The phase behaviour of a particular type of mucin is dependent on its molecular structure. In this work we visualised the molecular structure of porcine gastric mucin (PGM) in dehydrated state using the atomic force microscopy (AFM). Samples deposited on mica were studied in air by AFM operated in the tapping mode. Atomic force microscopy indicates the presence of a dumbbell structure (b, c, d) as well as fiberlike structure (a) at higher concentrations. Znamenskaya Y, Engblom J, Sotres J, Arnebrant T and Kocherbitov K. (To be published) a) b) c) d) Figure 4.5. Images of mucin structures obtained after evaporation of solutions. Initial mucin concentrations are: a) 10-3 %; b) 10-5 %; c) 10-5 %; d) 10-5 %, 3-D image of dumbbell mucin molecule 12

15 4.6 Differential effects of Pseudomonas aeruginosa on biofilm formation by fresh isolates and laboratory strains of Staphylococcus epidermidis Natural biofilms, such as plaque on teeth and the biofilms in the intestines consists of hundreds of different species. However, biofilms on medical implants are deprived of the rich flora and usually consist only of one or a few species. Pseudomonas aeruginosa and Staphylococcus epidermidis are common species found in infections related to medical implants. Studying dual species biofilms of P. aeruginosa and S. epidermidis, using 16S rrna FISH probes and confocal microscopy, we showed presence of P. aeruginosa to reduce biofilm formation of S. epidermidis. This inhibition was due to extracellular products of P. aeruginosa, possibly polysaccharides. Interestingly, different strains of S. epidermidis varied in their ability to withstand the inhibition of P. aeruginosa, with fresh isolates far exceeding laboratory strains. Maria Pihl, Julia R. Davies, Luis E. Chávez de Paz, Gunnel Svensäter. Differential effects of Pseudomonas aeruginosa on biofilm formation by fresh isolates and laboratory strains of Staphylococcus epidermidis. Submitted to FEMS Immunol Microbiol 5 µm Figure 4.6. The figure show 9 hour biofilms of two different S. epidermidis strains (Mia and C103) alone (a, c) and in dual-species biofilms (b, d) with P. aeruginosa NCTC 6750, clearly showing P. aeruginosa to be able to reduce biofilm formation of S. epidermidis. 13

16 4.7 The effects of antimicrobials in endodontic biofilm bacteria In the present study, confocal microscopy, a mini flow-cell system and image analysis were combined to test in situ the effect of antimicrobials and alkali on biofilms of Enterococcus faecalis, Lactobacillus paracasei, Streptococcus anginosus and Streptococcus gordonii isolated from root canals with persistent infections. Biofilms formed for 24h were exposed for 5 min to alkali (ph 12), chlorhexidine digluconate (2.5%), EDTA (50mM) and sodium hypochlorite (1%). The biofilms were then characterized using fluorescent markers targeting cell membrane integrity (LIVE/DEAD) and metabolic activity (CTC and FDA). In addition, the biofilm architecture and the extent to which coating of the substrate surface with collagen influenced the resistance pattern to the chemicals were also analyzed. NaOCl (1%) affected membrane integrity of all organisms and removed most biofilm cells. Exposure to EDTA (50mM) affected the membrane integrity in all organisms, but failed to remove more than a few cells in biofilms of E. faecalis, L. paracasei and S. anginosus. Chlorhexidine (2.5%) had a mild effect on the membrane integrity of E. faecalis and removed only 50% of its biofilm cells The effects were substratum-dependent and most organisms displayed increased resistance to the antimicrobials on collagen-coated surfaces. The biofilm system developed here was sensitive and differences in cell membrane integrity and removal of biofilm cells after exposure to antimicrobials commonly used in endodontics was discernable. Chávez de Paz LE, Bergenholtz G, Svensäter G. The effects of antimicrobials on endodontic biofilm bacteria.j Endod 2010;36:70-7. Figure 4.7. Three-dimensional reconstructions from confocal micrographs showing the effect of chlorhexidine and alkali on biofilms of Enterococcus faecalis and S. gordonii uing LIVE/DEAD staining. The green cells represent cells with intact membranes, wheras the red cells are damaged or dead. Images were constructed using the software bioimage_l. 14

17 4.8 A novel method to detect gene expressions in biofilms Microbial biofilms constitute a major medical problem when formed on human tissues due to their high resistance to an unlimited number of environmental stress including antibiotic and biocide treatments. This decreased susceptibility of biofilm-forming bacteria to host defenses and antibiotic treatments is related to a heterogeneous distribution of sub-populations of micro-organisms with altered patterns of gene expression. This biological heterogeneity in biofilm populations need to be studied by techniques which do not disrupt the natural distribution of biofilm cells. We have developed a fluorescense in situ hybridization (FISH) method to detect gene expression in situ at early stages of biofilm formation. In this protocol we use multiple probes targeting mrna sequences in mono and dual species cultures. As observed in Figure 4.8, probes targeting the gene expression of serine protease challisin in Streptococcus gordonii (yellow signals) were simultaneously visualized with 16S rrna probes that target Streptococcus gordonii (red cells) and Lactobacillus salivarius (blue cells). By using this combined protocol we could successfully monitored in situ gene expression in microbial biofilms. Luis Chávez de Paz, Julia Davies, Gunnel Svensäter (To be published) Figure 4.8. Probes targeting the gene expression of serine protease challisin in Streptococcus gordonii (yellow signals) were simultaneously visualized with 16S rrna probes that target Streptococcus gordonii (red cells) and Lactobacillus salivarius (blue cells). 15

18 4.9 Effect of fluoride and Lactobacillus rhamnosus on plaque acid tolerance Fluoride has long been used in preventive strategies against dental caries due to its hydroxyapatite incorporation that lower the solubility of the enamel and dentine. From in vitro-studies it is known that fluoride also affects the physiology of oral bacteria making them acid sensitive but data on the effect of acid tolerance in plaque in vivo is limited. The aim of the present investigation was to study the effect of fluoride and probiotic strain Lactobacillus rhamnosus LB21 on plaque acid tolerance and lactic acid production in vivo. Subjects participating in this study were instructed to drink milk containing fluoride or/and L. rhamnosus on a daily basis for 15 months. Plaque samples were taken at baseline and after 15 months. Plaque acid tolerance was measured using LIVE/DEAD BacLight TM Viability Stain after exposure to ph 3.5 for 2 hours. The results showed that dental plaque from subjects that had ingested milk containing fluoride or fluoride in combination with L. rhamnosus exhibited a significant decrease in acid tolerance after 15 months compared baseline (see Figure 4.9). Subjects that had been drinking milk without fluoride or with lactobacilli only did not show any decrease in acid tolerance. Lactic acid production from glucose was also lower in the groups that had been exposed to fluoride. BASELINE AFTER 15 MONTHS DECREASED PLAQUE ACID TOLERANCE INCREASED PLAQUE ACID TOLERANCE Figure 4.9. Examples on plaque acid tolerance in two different subjects visualized using LIVE/DEAD BacLight TM Viability stain after the exposure to ph 3.5 fro two hours. Conclusively, this pilot study shows that fluoride affects plaque acid tolerance and lactic acid production in vivo. L. rhamnosus on the other hand does not seem to affect plaque acid tolerance or lactic acid production in either direction. Welin-Neilands J, G. Svensäter G To be presented at the IADR congress in Barcelona,

19 4.10 Mucin-bacterial interactions degradation of MUC5B by dental biofilms Members of the indigenous oral microbiota must interact to degrade and utilize complex salivary components in order to survive and grow in the oral cavity. We have shown for the first time, that a naturally occurring substrate, MUC5B, is proteolytically digested preferentially by cooperation of several members of the dental plaque microbiota. Interspecies cooperation appears to overcome the high level of molecular complexity in MUC5B and makes the digestion products available as a nutrient source. Endopeptidase activities in conjunction with full or partial deglycosylation, may be the first step in the degradation of salivary proteins by the oral biofilm. This first step in degradation would increase the availability and number of N- and C-termini to serve as substrates for aminopeptidases, which liberate amino acids and short peptides to support bacterial growth. Mono-cultures was not able to degrade the protein backbone of the MUC5B mucin (a), but a consortia of four species efficiently degraded the protein (b). When incubated with pooled dental plaque, MUC5B was degraded less efficiently than with the four-species consortium (c). This illustrates the diversified functions of the polymicrobial species in dental plaque and that a balance is established between the multispecies biofilm and host glycoproteins, that is beneficial to oral health. Wickström, C., Herzberg, M. C., Beighton, D. & Svensäter, G. Proteolytic degradation of human salivary MUC5B by dental biofilms (2009) Microbiology 155, Figure SDS/PAGE Western blot showing degradation of the MUC5B polypeptide backbone. MUC5B was incubated with (a) Streptococcus gordonii (b) a four-species consortium containing Streptococcus gordonii, Steptococcus biovar 2, Streptococcus cristatus and Actinomyces naeslundii and (c) supragingival dental plaque. 17

20 4.11 Antibody binding kinetics in ELISA The purpose of an ELISA (Enzyme Linked Immunosorbent Assay) is usually to detect an antigen or antibody in a biological sample. In the detection of a specific antibody a surface pre-coated with the antigen is exposed to the sample. The binding is then detected by a second antibody that is covalently linked to an enzyme. The final step in the assay is to determine the enzymatic activity, which is a measure of the amount of the specific antibody in the sample. In this respect an ELISA can be regarded as a black box, where only the end result is obtained. Therefore, the objective of the study was to elucidate the possibilities to follow the binding kinetics with ellipsometry. This is an optical technique that does not require labeling of the molecules involved, which can be used to follow the mass and thickness of thin films on solid substrates in situ. The binding study was performed on commercial surfaces used for ELISA and the experiment followed the standard procedure that is used in this assay. In the figure below the thickness of the protein layer is presented during the complete ELISA procedure. The addition of the specific antibody did not result in an increase in the layer thickness. However, after the addition of the secondary antibody a clear increase was obtained proving the existence the specific antibody on the surface. Information on the binding kinetics may be useful to improve existing assays for specific antibodies with respect to incubation times and concentrations. Also, it can provide information on the role of other components such as blocking proteins and detergents that are used in these assays Thickness (nm) Antigen Specific antibody Secondary conjugated antibody Time (min) Figure Thickness of the protein layer versus time for an ELISA used to detect a specific antibody. Washing steps (not indicated) preceded the additions of the specific and secondary antibodies. 18

21 5 PhD Theses Supervised by Center Members 1. Olof Svensson, Malmö University (Supervisors Thomas Arnebrant and Krister Thuresson (Camurus AB)): Interactions of mucins with biopolymers and drug delivery particles ( ) 2. Jessica Neilands, Malmö University (Supervisors: Gunnel Svensäter and Thomas Arnebrant ) Acid tolerance of streptococcus mutans biofilms ( ) 3. Christer Spegél, Malmö University (Supervisors: Tautgirdas Ruzgas and Rafael Taboryski (Sophion Bioscience A/S)) Electrochemical monitoring of living cells ( ) 4. Erik Alpkvist, Malmö University (Supervisors: Anders Heyden and Niels Overgaard) Mathematical Modeling of Biofilms: Theory, Numerics and Applications ( ) 5. Ida Svendsen, Malmö University (Supervisors Thomas Arnebrant and Liselott Lindh):"In vitro and in vivo studies of salivary films at solid/liquid interfaces" ( ) 6. Karolina Haberska, Lund University (Supervisor Tautgirdas Ruzgas): " Protein and electrolyte layer-by layer films: Assembly and electron transfer ( ) 7. Linda Andersson, Malmö University (Supervisor Håkan Eriksson): Endocytosis by human dendritic cells ( ) 8. Peter Hellman, Malmö University (Supervisor Håkan Eriksson): Human dendritic cells- A study of early events during pathogen recognition and antigen endocytosis ( ) 9. Jildiz Hamit, Malmö University (Supervisors Thomas Arnebrant and Krister Eskilsson (Kemira AB)) "Interactions of adsorbed layers of carbohydrate containing polymers - Saliva, mucins and bacterial surfaces" (2005-, Lic thesis defended 2009) 10. Maria Pihl, Malmö University (Supervisors Gunnel Svensäter, Bertil Kinnby, Thomas Arnebrant) Biofilms on Peritoneal Dialysis Catheters (2005-) 11. Ulf Hejman, Malmö University (Supervisor Per Ståhle, co-supervisor Christina Bjerkén): "Biologivcally induced stress corrosion" (2005-) 12. Alma Masic, Lunds University (Supervisor Anders Heyden, co-supervisor Niels Chr. Overgaard) "Mathematical modeling of biofilms" (2007-) 13. Sebastian Björklund, Lund University (Supervisors Emma Sparr (LU), Johan Engblom (Mah) and Krister Thuresson (Camurus AB)): "Water - a crucial factor in regulating biomembrane permeability" (2008-) 14. Adnan Safdar, Lunds University (Supervisor Liu-Ying Wei, co-supervisor Per Ståhle) "Biocompatibility of ion beam melted materials" (2008-) 15. Anton Fagerström, Malmö University (Supervisors Johan Engblom, Vitaly Kocherbitov and Karin Bergström (AkzoNobel)): Bioavailability of active ingredients used in agriculture (2009-) 16. Yana Znamenskaya, Malmö University (Supervisors Vitaly Kocherbitov and Johan Engblom): Hydration of mucous gel (2009-) 19

22 17. Christian Alfredsson Kindblom, Malmö University (Supervisors Gunnel Svensäter, Claes Wickström, Madeleine Rohlin): Biofilm activity as a marker to identify patients at risk mechanisms underlying microbial stress tolerance (2009-) 18. Marjan Dorkhan, Malmö University (Supervisors Julia Davies, Gunnel Svensäter, Ann Wennerberg): Activities of microbial biofilms on bioactive implant surfaces (2009-) 19. Kostas Bougas, Malmö University (Supervisors Ann Wennerberg, Pentti Tengvall GU), Victoria Franke Stenport (GU)): Protein coat and bone incorporation (2009-) 20. Lory Melin, Malmö University (Supervisors Ann Wennerberg, Martin Andersson (Promimic AB): On the importance of nanometer structures for implant incorporation in bone tissue (2009-) 21. Magnus Falk, Malmö University (Supervisors Tautgirdas Ruzgas, Sergey Shleev): Three-dimensional nanobiostructure-based self-contained devices for biomedical application (2009-) 22. Rickard Hägglund, LTH (Supervisors P Ståhle, P Isaksson): Damage of paper materials (2009-) 23. Jon Lind, LTH (Supervisors A Massih, C Bjerkén): Methods for evaluation of evaluation of the hydride embrittlement of Ni-based super alloys (2009-) 24. Tuerdi Maymaytilli, LTH (Supervisors C Bjerkén, P Ståhle): Influence of plastic deformation on the formation and growth of embritteling metal hydride's" (2009-) 6 Collaborative Partners of the Center 6.1 Industry collaborators (* Partners within KKs-Biofilm grant) 1. ACO Hud Nordic AB 2. Akzo Nobel AB* 3. Anordica AB 4. AnoxKaldnes AB* 5. Arcam AB* 6. Arla Foods AB* 7. AstraZeneca R&D Lund* 8. Bioglan AB 9. Bioinvent International AB 10. Camurus AB* 11. Euro-Diagnostica AB* 12. Eviderm AB 13. Galenica AB 14. Gambro Lundia AB* 15. Genovis AB 16. InnoScandinavia AB 17. Medeon AB 18. Medicon Valley Alliance 19. Nares AB 20. Nobel Biocare AB 21. Novosense AB 22. Novozymes A/S, Denmark 23. Phase Holographic Imaging AB 24. Promimic AB* 25. PVA-MV AG, Germany 26. QuNano AB 27. Quantumwise A/S, Denmark 28. Sinclair Pharma AB* 29. Stora Enso AB 30. Studsvik Nuclear AB 31. TetraPak AB 32. Volvo Aero AB 33. YKI AB 20

23 6.2 Academic collaborators Collaborations with other universities and research institutions in Sweden 1. Prof. em. Kåre Larsson, Camurus Lipid Research Foundation,, Lund 2. Prof. Gunnar Bergenholtz, Microbiology/Endodontics University of Gothenburgh 3. Prof. Per Claesson, The Royal Institute of Technology (KTH) and Surface Chemistry Institute (YKI), Stockholm 4. Professor Gunnar Dahlén, Microbiology/Endodontics University of Gothenburgh 5. Prof. Lo Gorton, Biochemistry, Lund University 6. Prof. Christer Hansson, Dermatology, Lund University 7. Prof. Göran Lundborg, Hand Surgery, Malmö, Lund University 8. Prof. Martin Malmsten, Pharmacy, Uppsala University 9. Prof. Jan Nilsson, CRC UMAS, Lund University 10. Prof. Tommy Nylander, Physical Chemistry 1, Lund University 11. Prof. Adrian Rennie, Physics, Uppsala University 12. Prof. Mark Rutland, The Royal Institute of Technology (KTH) and Surface Chemistry Institute (YKI), Stockholm 13. Prof. Olle Söderman, Physical Chemistry 1, Lund University 14. Prof. Per Uvdal, MAX lab, Lund University 15. Prof. Martin Andersson, Dept Applied Chemistry, Chalmers university of Technology. 16. Prof. Pentti Tengvall, Dept Biomaterials, Sahlgrenska Academy, Göteborg University 17. Prof. Tomas Albrektsson, Dept Biomaterials, Sahlgrenska Academy, Göteborg University 18. Prof Marie Wahlgren, Food Technology, LTH, Lund University 19. Prof. Ingegerd Johansson, Cariology, Umeå University 20. Assoc. Prof. Birgitta Rosén, Hand Surgery, Malmö, Lund University 21. Assoc. Prof. Lars Norlén, Cell- and Molecular biology, Karolinska Institute 22. Assoc. Prof. Viveka Alfredsson, Physical Chemistry 1, Lund University 23. Assoc. Prof. Ola Bergendorff, Dermatology, Lund University 24. Assoc. Prof. Emma Sparr, Physical Chemistry 1, Lund University 25. Assoc.Prof. Peter Siesjö, Department of Clinical Sciences, BMC, Lund University 26. Assoc. Prof.Eva Blomberg, The Royal Institute of Technology (KTH) and Surface Chemistry Institute (YKI), Stockholm 27. Associate Professor Victoria Franke-Stenport, Dept Prosthodontics, Sahlgrenska Academy, Göteborg University 28. Dr Yngve Cerenius, MAX lab, Lund University 29. Dr Adam Feiler, The Royal Institute of Technology (KTH) and Surface Chemistry Institute (YKI), Stockholm 30. Dr Anders Björkman, Hand Surgery, Malmö, Lund University 31. Dr Isabel Goncalves, CRC UMAS, Lund University 32. Dr Lars G Petersson, Specialist Clinic for Oral Health Care, Hallands Läns Landsting, Halmstad 33. Dr. Justas Barauskas, Institute of Biochemistry, Vilnius, Lithuania 34. Dr Robert Corkery, The Royal Institute of Technology (KTH) and Surface Chemistry Institute (YKI), Stockholm 35. Dr Anna Westerlund, Odontology, Gothenburg University 36. Dr. Ivan Maximov, Solid State Physics, Lund University 37. Dr. Johan Drott, Lund University Innovation, Lund University 38. Dr Valera Veryazov, Theoretical Chemistry, Lund University 21

24 International collaborations 1. Prof. em. Ian Hamilton, University of Manitoba, Dept of Oral Biology, Winnipeg, Canada 2. Prof. Robert Baier, University of Buffalo, Industry/University Cooperative Research Center for Biosurfaces, Buffalo, USA 3. Prof. F. C. Shih, Singapore National University, Singapore 4. Prof. Leslie Banks-Sills, Cornell University, Ithaca, NY, USA and University of Tel Aviv, Israel 5. Prof. Iwona Beech, University of Portsmouth, UK 6. Prof. Jan Sunner, University of Portsmouth, UK 7. Prof. David Beighton, Guy's Kings and St Thomas' Dental Institute, Joint Microbiology Research Unit, London, UK 8. Prof. R. Singh, Indian Institute of Technology, Mumbai, India 9. Prof. S. Surresh, MIT, Cambridge, Materials Science, USA 10. Prof. Bent Sörensen, Denmark Technical University, Riso Labs, Denmark 11. Prof. R. K. Thomas, Oxford University, Dept of Physical Chemistry, and ISIS at RAL (Neutron reflection facility), Oxford 12. Prof. Regine Willumeit, GKSS Research Centre, Geesthacht, Germany 13. Prof. Mark Hertzberg, University of Minnesota, Department of Microbiology, Minneapolis, USA 14. Prof. A. Needleman, Brown university, Providence, USA 15. Prof. Jukka Meurman, Helsinki University Central Hospital, Dept of Oral and Maxillofacial Diseases, Helsinki, Finland 16. Prof. Christopher Exley, Keele University, UK 17. Prof. Takashi Sawase, Dept Prosthodontics, University of Nagasaki, Japan. 18. Prof. Wolfgang Schuhmann, Ruhr-Universität Bochum, Germany 19. Prof. Edmond Magner, University of Limerick, Ireland 20. Prof. Dietmar Haltrich, Universität für Bodenkultur Wien, Austria 21. Prof. Phil Bartlett, The University of Southampton, UK 22. Assoc. Prof. Dennis Cvitkovitch, University of Toronto, Dept of Microbiology, Toronto, Canada 23. Assoc. Prof. Jeannine Brady, University of Florida, Dept of Oral Biology, Gainesville, USA 24. Assoc. Prof. Marie Ranson, School of Biological Sciences, Scientific Director Cancer, Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, Australia 25. Assoc. Prof. Kamal Mustafa, Bergen University, Bergen, Norway. 26. Assoc. Prof. Duncan Sutherland, Aarhus University, Denmark 27. Dr Michael Ortize, Caltech, USA 28. Dr Andrey Jivkov, Manchester University, UMIST, UK 29. Dr. Isaac Klapper, Montana State University, Center of Biofilms Research, Montana, USA 30. Dr Juozas Kulys, Inst. of Biochemistry, Vilnius, Lithuania 31. Dr. Anne Meyer, University of Buffalo, Industry/University Cooperative Research Center for Biosurfaces, Buffalo, USA 32. Dr Srikumar Banerjee, Indian Atomic Research Centre, India 33. Dr. G. Fragneto, ILL (Neutron reflection facility), Grenoble 34. Dr Adam Heller, University of Texas at Austin, TX, USA 35. Dr Kenneth Holmberg, Tekniska högskolan i Helsinki, Finland 36. Dr Rafael Taborisky, Denmark Technical University, Riso Labs, Denmark 37. Dr Alexander Yaropolov, Inst. of Biochemistry, Moscow 38. Dr. Sergei Lobov, School of Biological Sciences, University of Wollongong, Wollongong, Australia 39. Dr. Miguel Alcalde, Consejo Superior de Investigaciones Científicas, Applied Biocatalysis group, Spain 40. Dr. Antonio L. De Lacey,, Consejo Superior de Investigaciones Científicas, Bioelectrocatalysis group, Spain 41. Dr. Donal Leech, National University of Ireland, Galway, Ireland 22

25 7 List of Publications of the Center from 2009 and Onwards All titles listed under journal articles, review papers, and book chapters have been or are subjected to peer review. Center publications for the period can be found at Journal articles and invited review papers in journals 1. Chávez de Paz LE, Bergenholtz G, Svensäter G. The effects of antimicrobials on endodontic biofilm bacteria.j Endod 36 (2010) Chavez de Paz LE. Image analysis software based on color segmentation for characterization of viability and physiological activity of biofilms. Appl Environ Microbiol 75 (2009) Davies J, Svensäter G, Herzberg MC. Identification of novel surface proteins from Streptococcus gordonii involved in adhesion to oral surfaces. Microbiology 155 (2009) Wickström C, Herzberg MC, Beighton D, Svensäter G. Proteolytic degradation of human salivary MUC5B by dental biofilms. Microbiology 155 (2009) Wickström C, Hamilton I, Svensäter G. Differential metabolic activity by dental plaque bacteria in association with two preparations of MUC5B mucins in solution and in biofilms. Microbiology 155 (2009) Holmberg K, Ronkainen H, Laukkanen A, Wallin K, Hogmark S, Jacobson S, Wiklund S, Wiklund U, Souza RM, Ståhle P. Residual stresses in TiN, DLC and MoS2 coated surfaces with regard to their tribological fracture behaviour Journal of Wear (2009) Singh RN, Ståhle P, Chakravartty JK, Shmakov AA. Threshold stress intensity factor for delayed hydride cracking in Zr-2.5%Nb pressure tube alloy. Materials Science and Engineering a-structural Materials Properties Microstructure and Processing, 523 (2009) Massih AR, Diffusion-controlled phase growth on dislocations, Philosophical Magazine, 89:33 (2009) Massih AR, Jernkvist LO, Stress orientation of second-phase in alloys: Hydrides in zirconium alloys, Computational Materials Science, 46:4 (2009) Massih AR, Jernkvist LO, Transformation kinetics of alloys under non-isothermal conditions, Modelling and Simulation in Materials Science and Engineering, 17:5 (2009) 11. Massih AR, Transformation kinetics of zirconium alloys under non-isothermal conditions, Journal of Nuclear Materials, 384:3 (2009) Hellman P, Andersson L, Eriksson H. Ligand surface density is important for efficient capture of immunoglobulin and phosphatidylcholine coated particles by human peripheral dendritic cells. Cellular Immunology, 258 (2009) Gauffin F, Diffner E, Gustafsson B, Nordgren E, Gjörloff Wingren A, Sander B, Liao Persson J Gustafsson B. Expression of PTEN and SHP1, investigated from tissue micro arrays in pediatric acute lymphoblastic leukaemia. Pediatric Hematology and Oncology, 26 (2009) Göransson A, Arvidsson A, Currie F, Franke-Stenport V, Kjellin P, Mustafa K, Sul T, Wennerberg A. An in vitro comparison of possibly bioactive titanium implant surfaces. J Biomed Mater Res A 15:88(4) (2009) Eliasson A, Blomqvist F, Wennerberg A, Johansson A. A Retrospective Analysis of Early and Delayed Loading of Full-Arch Mandibular Prostheses Using Three Different Implant Systems: Clinical Results with Up to 5 Years of Loading. Clin Implant Rel Res 11:2 (2009) Wennerberg A, Albrektsson T. Effects of titanium surface topography on bone integration. Review Clin Oral Impl Research 20(suppl 4) (2009) Wennerberg A, Fröjd V, Olsson M, Nannmark U, Emanuelsson L, Johansson P, Josefsson Y, Kangasniemi I, Peltola T, Tirri Teemu, Pänkäläinen T, Thomsen P. Nanoporous TiO2 thin film on titanium oral implants for enhanced human soft tissue contact. A light and electron microscopy study. Clin Impl Dent Rel Res (2009) 23