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LORY MELIN SVANBORGON THE IMPORTANCE OF NANOMETER STRUCTURES FOR IMPLANT INCORPORATION IN BONE TISSUE

O n t h e i m p O r t a n c e O f n a n O m e t e r s t r u c t u r e s f O r i m p l a n t i n c O r p O r a t i O n i n b O n e t i s s u e

Malmö UniversityFaculty of Odontology Doctoral Dissertations 2011

© Lory Melin Svanborg 2011

ISBN 978-91-7104-384-9

Holmbergs, Malmö 2011

lOry melin svanbOrg On the impOrtance Of nanOmeter structures fOr implant incOrpOratiOn in bOne tissue

Department of Prosthodontics,Faculty of Odontology at Malmo University

Department of BiomaterialsInstitute of Clinical Sciences

The Sahlgrenska Academy at University of Gothenburg2011

Publikationen finns även elektroniskt,se www.mah.se/muep

To my family and friends, with love.

Life is not the days that have passed, but the days you remember –

Carpe Diem...

This thesis represents number 41 in a series of investigations on implants, hard tissue and the locomotor apparatus originating from the department of Biomaterials/Handicap Research, University of Gothenburg and the department of Prosthodontics, Malmö University, Sweden.

1. Anders R Eriksson DDS, 1984. Heat-induced Bone Tissue Injury. An in vivo

investigation of heat tolerance of bone tissue and temperature rise in the drilling of

cortical bone. Thesis defended 21.2.1984. External examiner: Docent K-G. Thorngren.

2. Magnus Jacobsson MD, 1985. On Bone Behaviour after Irradiation. Thesis

defended 29.4.1985. External examiner: Docent A. Nathanson.

3. Fredrik Buch MD, 1985. On Electrical Stimulation of Bone Tissue. Thesis defended

28.5.1985. External examiner: Docent T. Ejsing-Jörgensen.

4. Peter Kälebo MD, 1987. On Experimental Bone Regeneration in Titanium Implants.

A quantitative microradiographic and histologic investigation using the Bone Harvest

Chamber. Thesis defended 1.10.1987. External examiner: Docent N. Egund.

5. Lars Carlsson MD, 1989. On the Development of a new Concept for Orthopaedic

Implant Fixation. Thesis defended 2.12.1989. External examiner: Docent L-Å Broström.

6. Tord Röstlund MD, 1990. On the Development of a New Arthroplasty. Thesis

defended 19.1.1990. External examiner: Docent Å. Carlsson.

7. Carina Johansson Techn Res, 1991. On Tissue Reactions to Metal Implants. Thesis

defended 12.4.1991. External examiner: Professor K. Nilner.

8. Lars Sennerby DDS, 1991. On the Bone Tissue Response to Titanium Implants.

Thesis defended 24.9.1991. External examiner: Dr J.E. Davis.

9. Per Morberg MD, 1991. On Bone Tissue Reactions to Acrylic Cement. Thesis

defended 19.12.1991. External examiner: Docent K. Obrant.

10. Ulla Myhr PT, 1994. On Factors of Importance for Sitting in Children with Cerebral

Palsy. Thesis defended 15.4.1994. External examiner: Docent K. Harms-Ringdahl.

11. Magnus Gottlander MD, 1994. On Hard Tissue Reactions to Hydroxyapatite-

Coated Titanium Implants. Thesis defended 25.11.1994. External examiner: Docent P.

Aspenberg.

12. Edward Ebramzadeh MScEng, 1995. On Factors Affecting Long-Term Outcome

of Total Hip Replacements. Thesis defended 6.2.1995. External examiner: Docent L.

Linder.

13. Patricia Campbell BA, 1995. On Aseptic Loosening in Total Hip Replacement:

the Role of UHMWPE Wear Particles. Thesis defended 7.2.1995. External examiner:

Professor D. Howie.

14. Ann Wennerberg DDS, 1996. On Surface Roughness and Implant Incorporation.

Thesis defended 19.4.1996. External examiner: Professor P.-O. Glantz.

15. Neil Meredith BDS MSc FDS RCS, 1997. On the Clinical Measurement of Implant

Stability and Osseointegration. Thesis defended 3.6.1997. External examiner: Professor

J. Brunski.

16. Lars Rasmusson DDS, 1998. On Implant Integration in Membrane-Induced and

Grafter Bone. Thesis defended 4.12.1998. External examiner: Professor R. Haanaes.

17. Thay Q Lee MSc, 1999. On the Biomechanics of the Patellofemoral Joint and

Patellar Resurfacing in Total Knee Arthroplasty. Thesis defended 19.4.1999. External

examiner: Docent G. Nemeth.

18. Anna Karin Lundgren DDS, 1999. On Factors Influencing Guided Regeneration

and Augmentation of Intramembraneous Bone. Thesis defended 7.5.1999. External

examiner: Professor B. Klinge.

19. Carl-Johan Ivanoff DDS, 1999. On Surgical and Implant Related Factors

Influencing Integration and Function of Titanium Implants. Experimental and Clinical

Aspects. Thesis defended 12.5.1999. External examiner: Professor B. Rosenquist.

20. Bertil Friberg DDS MDS, 1999. On Bone Quality and Implant Stability

Measurements. Thesis defended 12.11.1999. External examiner:

Docent P. Åstrand.

21. Åse Allansdotter Johnsson MD, 1999. On Implant Integration in Irradiated

Bone. An Experimental Study of the Effects of Hyberbaric Oxygenation and Delayed

Implant Placement. Thesis defended 8.12.1999. External examiner: Docent K. Arvidsson-

Fyrberg.

22. Börje Svensson DDS, 2000. On Costochondral Grafts Replacing Mandibular

Condyles in Juvenile Chronic Arthritis. A Clinical, Histologic and Experimental Study.

Thesis defended 22.5.2000. External examiner: Professor Ch. Lindqvist.

23. Warren Macdonald BEng, MPhil, 2000. On Component Integration in Total Hip

Arthroplasty: Pre-Clinical Evaluations. Thesis defended 1.9.2000. External examiner: Dr

A.J.C. Lee.

24. Magne Røkkum MD, 2001. On Late Complications with HA Coated Hip

Asthroplasties. Thesis defended 12.10.2001. External examiner: Professor P. Benum.

25. Carin Hallgren Höstner DDS, 2001. On the Bone Response to Different Implant

Textures. A 3D analysis of roughness, wavelength and surface pattern of experimental

implants. Thesis defended 9.11.2001. External examiner: Professor S. Lundgren.

26. Young-Taeg Sul DDS, 2002. On the Bone Response to Oxidised Titanium

Implants: The role of microporous structure and chemical composition of the surface oxide

in enhanced osseointegration. Thesis defended 7.6.2002. External examiner: Professor

J.-E. Ellingsen.

27. Victoria Franke Stenport DDS, 2002. On Growth Factors and Titanium Implant

Integration in Bone. Thesis defended 11.6.2002. External examiner: Associate Professor

E. Solheim.

28. Mikael Sundfeldt MD, 2002. On the Aetiology of Aseptic Loosening in Joint

Arthroplasties and Routes to Improved cemented Fixation. Thesis defended 14.6.2002.

External examiner: Professor N Dahlén.

29. Christer Slotte DDS, 2003. On Surgical Techniques to Increase Bone Density

and Volume. Studies in the Rat and the Rabbit. Thesis defended 13.6.2003. External

examiner: Professor C.H.F. Hämmerle.

30. Anna Arvidsson MSc, 2003. On Surface Mediated Interactions Related to

Chemo-mechanical Caries Removal. Effects on surrounding tissues and materials. Thesis

defended 28.11.2003. External examiner: Professor P. Tengvall.

31. Pia Bolind DDS, 2004. On 606 retrieved oral and cranio-facial implants. An

analysis of consecutively received human specimens. Thesis defended 17.12. 2004.

External examiner: Professor A. Piattelli.

32. Patricia Miranda Burgos DDS, 2006. On the influence of micro-and macroscopic

surface modifications on bone integration of titanium implants. Thesis defended 1.9.

2006. External examiner: Professor A. Piattelli.

33. Jonas P Becktor DDS, 2006. On factors influencing the outcome of various

techniques using endosseous implants for reconstruction of the atrophic edentulous and

partially dentate maxilla. Thesis defended 17.11.2006. External examiner: Professor K.

F. Moos

34. Anna Göransson DDS, 2006. On Possibly Bioactive CP Titanium Surfaces. Thesis

defended 8.12.2006 External examiner: B. Melsen

35. Andreas Thor DDS, 2006. On platelet-rich plasma in reconstructive dental implant

surgery. Thesis defended 8.12.2006. External examiner: Prof E.M. Pinholt.

36. Luiz Meirelles DDS MSc, 2007. On Nano Size Structures For Enhanced Early Bone

Formation. Thesis defended 13.6.2007. External examiner: Professor Lyndon F. Cooper.

37. Pär-Olov Östman DDS, 2007. On various protocols for direct loading of implant-

supported fixed prostheses. Thesis defended 21.12.2007. External examiner: Prof B

Klinge

38. Kerstin Fischer DDS, 2008. On immediate/early loading of implant supported

prostheses in the maxilla. Thesis defended 8.2.2008. External examiner: Professor

Kristina Arvidson Fyrberg

39. Alf Eliasson 2008. On the role of number of fixtures, surgical technique and timing

of loading. Thesis defended 23.5.2008. External examiner: Kristina Arvidson-Fyrberg.

40. Victoria Fröjd DDS, 2010. On Ca2+ incorporation and nanoporosity of titanium

surfaces and the effect on implant performance. Thesis defended 26.11.2010. External

examiner: Professor J. E. Ellingsen

41. Lory Melin Svanborg DDS, 2011. On the importance of nanometer structures

for implant incorporation in bone tissue. Thesis to be defended 01.06.2011. External

examiner: Associate professor Christer Dahlin

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Ontheimportanceofnanometer

structuresfor implantincorporationinbonetissue

Lory Melin Svanborg

Department of Prosthodontics, Faculty of Odontology at Malmo University

Department of Biomaterials Institute of Clinical Sciences

The Sahlgrenska Academy at University of Gothenburg

2011

On the importance of nanometer structures for

implant incorporation in bone tissue

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abstract

introductionReplacing lost teeth with dental implants is today a reliable treatment method associated with good long-term clinical results. Different surface modifications alter the surface topography at micro- and nanometer level of resolution as well as chemical properties, which have shown to be of importance for osseointegration. Research within the field of implantology is still intense and aims at further improving the implant properties to achieve successful treatments for patients with compromised bone as well as developing a surface that provides faster integration to shorten the treatment period. Furthermore, more basic science data is needed to increase our understanding of the mechanisms involved in osseointegration.

The significance of the surface topography on the micrometer level for implant integration is well known. However, the knowledge of how and to what extent nanostructures may be of importance in early bonehealing and osseointegration remains to be investigated.

aimThe overall aim of this thesis was to describe a technique to characterize commercial oral implants on the nanometer level when nanostructures are applied on a microroughness and to investigate whether or not the nanometer surface roughness was correlated to the more well-known micrometer roughnesss; to study the real-time initial cellular interactions of human osteoblasts and fibroblasts to different implant surfaces with and without a coat of nanocrystalline hydroxyapatite; and to evaluate the early bone response to a nanocrystalline hydroxyapatite coating (nano-HA) applied on smooth cylindrical and moderately rough screw-shaped implants.

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materials and methodsTwelve different commercial screw-shaped dental implants with different surface modifications were examined using optical interferometry together with Gaussian digital filters and scanning electron microscopy (SEM). Human osteoblasts and fibroblasts were used when investigating the initial cell-surface interaction to different surfaces modifications with optical tweezers (OT) and quartz crystal balance with dissipation monitoring (QCM-D). To evaluate the effect of nanocrystalline hydroxyapatite (nano-HA) compared to nanosized particles of titanium in early bone response, smooth cylindrical titanium implants with no microroughness were inserted in rabbit tibia. The implant surfaces were examined using atomic force microscopy (AFM) and interferometry. To evaluate the biological response, histological analyses including bone contact (BIC) and bone area (BA), as well as qualitative analysis were performed. Furthermore, screw-shaped sandblasted and acid etched titanium implants coated with nano-HA of different thicknesses and un-coated controls were evaluated in rabbit tibia as well as femur. Interferometry, SEM and X-ray Photoelectron Spectroscopy (XPS) were used to characterize the implant surface topography and chemical composition. Biomechanical and histological evaluations including BA, newly formed bone and qualitative evaluations were performed.

results The studies showed that it is possible to characterize the surface nanoroughness of commercially dental implants using interferometry. A 1x1µm Gaussian filter was found useful to identify nanoroughness in terms of height deviation. It was demonstrated that the implants do have distinct roughness on the nanometer level of resolution and that the nanoroughness is not correlated to the microroughness when comparing mean surface roughness (Sa). Significant differences in Sa on the nanometer scale were found among some of the implants investigated. However, to detect specific nanostructures an additional SEM examination is necessary. The results from optical trap experiments showed that both osteoblasts and fibroblasts responded in a similar way towards most of the surfaces.

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No difference in initial cell attachment could be detected between the surfaces when using the QCM-D technique.

A nano-HA coating applied on smooth cylindrical implants did not enhance bone responses in terms of bone contact (BIC) and bone area (BA) values as compared to nano-titania.

Screw-shaped sandblasted and acid etched titanium implants with applied nanothick (~20nm) coating of nano-HA with similar Sa values on both micro- and nanometer scale of resolution presented similar removal torque values, BA values and showed similar amounts of newly formed bone as compared to un-coated controls when placed in cortical bone. The same result was demonstrated in trabecular bone with a submicron thick coating of nano-HA onto sandblasted and acid etched screw-shaped implants.

conclusionsWithin the limits of the studies in this thesis, it was demonstrated that commercially available oral implants do have nanoroughness of various amounts and that the nanoroughness is not correlated to the microroughness.

It was demonstrated possible to observe cell attachment using optical trapping and QCM-D, however no obvious differences between the surfaces could be detected.

A nano-HA coating applied on cylindrical titanium implants did not enhance early bone response compared to a nano-titania coating when evaluated in cortical bone. Furthermore, sandblasted and acid etched screw-shaped implants with applied coatings of different thicknesses of nano-HA perform similar as un-coated controls when evaluated in cortical and trabecular bone.

Correspondence:Lory Melin Svanborg, Department of Prosthodontics, Faculty of Odontology, Malmö University. Sweden.E-mail: lory.svanborg@mah.se

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this thesis is based on the following five papers:

Surface characterization;

I. “Surface characterization of commercial oral implants on the nanometer level.” Svanborg LM, Andersson M, Wennerberg A. J Biomed Mater Res B Appl Biomater. 2010 Feb;92(2):462-9.

In vitro study;

II. “Using optical tweezers and QCM-D to investigate initial human osteoblast and fibroblast attraction/adhesion towards different implant surfaces” Svanborg LM, Westas E, Wang L, Duran R, Mustafa K, Wennerberg A, Andersson M. In manuscript.

In vivo studies;

III. “Effect of hydroxyapatite and titania nanostructures on early in vivo bone response.” Meirelles L, Melin L, Peltola P, Kjellin P, Kangasniemi I, Currie F, Andersson M, Albrektsson T, Wennerberg A. Clin Implant Dent Relat Res. 2008 Dec;10(4):245-54.*

IV. “The effect of hydroxyapatite nanocrystals on early boneformation surrounding dental implants.” Svanborg LM, Hoffman M, Andersson M, Currie F, Kjellin P, Wennerberg A. Int J Oral Maxillofac Surg. 2011 Mar;40(3):308-15. Epub 2010 Dec 15.

V. “Evaluation of early bone healing on sandblasted and acid etched implants coated with nanocrystalline hydroxyapatite - An in vivo study in rabbit femur” Svanborg LM, Meirelles L, Franke-Stenport V, Kjellin P, Currie F, Andersson M, Wennerberg A. Submitted.

* This paper has previously appeared in a Ph D thesis by Luiz Meirelles (2007). L melin participated in surgery of animals, as well as in surface and histological analyses.

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table Of cOntents

ABSTRACT .................................................................. 11INTRODUCTION .......................................................... 17

Background and need for further studies on implant surfaces .....17Implant material, design and surface properties .......................18Microroughness ....................................................................21

Methods to alter microtopography ...................................21Nanoroughness and nanostructures ........................................25

Some methods to alter nanotopography ..............................26Nanostructures and biological response .............................29New possibilities to evaluate cell response to nanostructures .31

Summary and relevance of the present thesis ...........................32AIMS ......................................................................... 34MATERIALS AND METHODS ........................................... 35

Implants and components ......................................................35Implant design and surface preparation ..............................35

Methods for surface characterization ......................................36Atomic Force Microscope ................................................36Optical interferometry ......................................................37Gaussian filter and surface parameters ...............................37Scanning Electron Microscopy ...........................................39X-ray Photoelectron Spectroscopy ......................................39Powder X-ray Diffraction (PXRD) .........................................39

Methods for evaluation in vitro ...............................................40Cells and culture ..............................................................40Optical trapping ..............................................................40Quartz Crystal Microbalance with dissipation monitoring (QCM-D) ........................................42

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Methods for evaluation in vivo ..............................................42Animals and surgical technique .........................................42Removal torque analysis ...................................................43Histological specimen preparation and evaluation ...............43

Data analysis/statistics ..........................................................44RESULTS ..................................................................... 45

Surface characterisation ........................................................45SEM..... ..........................................................................45Interferometry characterization ..........................................46AFM........ .......................................................................48XPS and PXRD .................................................................48

Evaluation in vitro .................................................................49Optical trap ....................................................................49QCM-D ..........................................................................49

Evaluation in vivo .................................................................52Removal torque analysis ...................................................52Histological evaluation .....................................................52

DISCUSSION ............................................................... 53Implant material, design and surface characteristics ..................53Evaluation of surface characteristics ........................................53

AFM....... ........................................................................53SEM and XPS ..................................................................54Optical interferometry ......................................................54Gaussian filter and surface parameters ...............................54

Methods and results in vitro ...................................................55Optical trap ....................................................................56QCM-D ..........................................................................56

Methods and results in vivo ....................................................58Animals and surgical technique .........................................58Removal torque analysis ...................................................58Histological specimen preparation and evaluation ...............59

Summary and topics for future research ...................................61CONCLUSIONS ........................................................... 63POPULäRVETENSKAPLIG SAMMANFATTNING .................. 65ACKNOWLEDGEMENTS ............................................... 68REFERENCES ............................................................... 71STUDY I-V ................................................................... 85

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intrODuctiOn

background and need for further studies on implant surfaces“Osseointegration is a process whereby clinically asymptomatic rigid fixation of an alloplastic material is achieved and maintained in bone during loading”. This definition of osseointegration based on stability instead of histology was suggested by Zarb & Albrektsson 19911. Implant treatment should aim to achieve predictable clinical results with controlled clinical implants to ensure an adequate treatment for the patients with the goal to restore function and to the greatest possible extent, aesthetics.

Titanium has been used as an implant material since the discovery of osseointegration in the 1960´s by Professor Per-Ingvar Brånemark and others. Titanium implants have since then been used in different applications, for example; amputation prostheses, hip replacements, pacemakers and oral implants. Dental implants have become a common, often uncomplicated treatment method to replace lost teeth. Today, many millions of oral implants are placed in patients over the world annually. High clinical success rates, commonly over 90% have been reported for different implant surfaces after more than 10 years in function2-5. The mandible is often associated with higher success rates than the maxilla due to greater variations of bone quality in the latter. Albrektsson et al.6 proposed criteria for implant success to be absence of pain, infection, neuropathy, implant mobility and radiographic peri-implant radiolucency. Furthermore, no more than 1mm bone loss during the first year after insertion was accepted and thereafter less than 0.2mm bone loss annually. During the years, techniques have been developed to improve the

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implants, with respect to design and surface properties, and thereby the success rate has increased. However, many patients with poor bone quality and or quantity would benefit from improved implant designs and/or specific surface modifications. Six factors have previously been suggested to be of importance for osseointegration; the biocompatibility of the implant material, the implant macro design, the implant surface properties, the status of the bone (quality, quantity and health), surgical technique and implant loading conditions7. It has been demonstrated possible to promote implant incorporation in bone by altering surface properties such as; physical (e.g. energy, wettability), chemical (e.g. ion incorporation), mechanical (e.g. hardness, yield strength) and structure (topography, geometry, roughness) of the implants 8-13. The research within the field of biomaterials and implantology is still intense and aims at further improving oral implants. Lately, there has been a growing interest in how the presence of nanometer structures on an implant surface influences the early bone healing process and the subsequent osseointegration.

This thesis will concentrate on surface topography with particular direction towards the potential importance of nanometer topography for enhanced implant incorporation in bone tissue.

implant material, design and surface propertiesTitanium is the most commonly used material for bone integrated implants due to its excellent biocompatibility. According to the Oxford Dictionary of National Biography, titanium was first discovered by William Gregor in 1791. This metallic element is the 9th most common element in the earth crust and titanium may be extracted from the minerals rutile (TiO2) and ilmenite (FeTiO3). Rutile is the most natural and common form of TiO2 and contains iron, niobium and tantalum. Anatase and brookite are two more rare forms of TiO2. There are four grades of commercially pure titanium (c.p. Titanium) which consists of approximately 99 weight percent of pure titanium but differ in the amount of oxygen, iron, carbon nitrogen and hydrogen. The difference in concentration has a significant impact on the physical and mechanical properties; for example titanium have high strength-to-weight ratio, high melting

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point and low electrical and thermal conductivity. Titanium forms a very stable surface oxide within nanoseconds when exposed to air, which makes the metal highly chemical and corrosion resistant. This is often claimed to be the reason for the good biocompatible properties of titanium. The native oxide is 2-6 nm thick and consists mainly of TiO2. C.p. Titanium exists in two different crystal structures; hexagonal α-structure and a cubic β-structure. The former is prevalent in room temperature while the latter is formed by heating to 883˚C or more. To maintain the β-structure at room temperature, alloy elements are needed. Titanium alloys may contain both α- and β-structure, such as Ti-6Al-4V (titanium grade 5), which has good mechanical properties as well as adequate toughness and good corrosion resistance14,15. It has been reported that titanium alloys present similar biological response as to c.p. titanium16. In contrast, previous in vivo studies have demonstrated c.p. titanium to have significantly increased bone response as compared to titanium alloy samples17-20. However, human studies on titanium grade 5 implants (Ti-6Al-4V) have shown good clinical results, indicating good biocompatibility21,22. Implants manufactured from zirconia ceramics have demonstrated to be biocompatible, showing similar biological response as titanium and to have mechanical properties that make them suitable as materials for dental implants23,24. Most commercial dental implants available today are still manufactured from c.p. titanium grade 4.

It was suggested that the inferior bone response to the titanium alloy was due to leakage of aluminum ions. Ion release from different implant surfaces have previously been reported in the literature, however, not in toxic concentrations25,26. For example, Wennerberg et al.27 investigated if there was a correlation between surface roughness and ion release in vitro but found no such differences between the smoothest and roughest surface. However, in vivo, increased amounts of titanium were observed in the peri-implant bone after 1 year of implantation compared to after 12 weeks. Further, ion leakage from nanosized hydroxyapatite crystal (nano-HA) coated surface have been reported when inserted in rat. The released ions were shown to be eliminated from the body via blood and liver and the authors concluded that the nano-HA coating had a low probability to be a biological risk factor28.

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Surface properties have been shown to be of importance to alter bone responses and establish adequate osseointegration. Since the tissue mainly interacts with the outermost anatomic layers of the implant surface29, the surface may be of greater importance than the bulk material characteristics. There are many different ways of modifying the implant surface and thereby changing the surface topography and/or chemistry. Commonly, it is difficult to change one surface characteristic without affecting the others.

Some techniques, for example; plasma spraying, coatings and ion deposition, will add material to the implant surface. In contrast, there are techniques that will remove material from the surface e.g.; blasting, etching and oxidation. Surface modification is further created by a combination of different techniques. To be able to properly evaluate the effect of the surface properties in bone healing, the surfaces must be characterized in a standardized manner. Topography as well as chemistry must be described.

Surface topography is scale dependent; what may be considered a rough surface on a container ship is certainly not equal to what is considered rough on a dental implant. Overall, surface roughness is measurable on the mm, µm and nm length scales. Even picometer roughness may exist, however, its possible importance for implant take being beyond our present knowledge. For dental implants, mm roughness represents the design of the implant (i.e. overall shape and thread design); µm surface roughness is known to influence osseointegration whereas the precise importance of the nanometer roughness remains to be investigated.

Different implant designs have been tested in the treatment of edentulous patients. Threaded implants have proven superior to cylindrical designs in long-term studies30,31, due to improved load distribution32. Therefore, threaded screw shaped implants dominate the commercial market today. Different thread designs and implants with micro-threads with the aim to preserve the marginal bone level are commercially available33.

As mentioned, micrometer roughness is well-known and several in vitro, in vivo as well as human studies have shown that surface roughness have an impact on osseointegration and biomechanical fixation9,11,34-36. However, changes made on surface roughness at

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the micrometer level may simultaneously, if accidentally, result in changes at the nanometer level; hence it may be difficult to separate the importance of micro- and nanoroughness for implant integration.

microroughnessOn the micrometer level of resolution, dental implants may be divided into groups by the average surface roughness (S)a; smooth (Sa <0.5µm), minimally rough (Sa 0.5<1µm), moderately rough (Sa 1-2µm) and rough (Sa >2µm), as suggested by Wennerberg and Albrekstsson 200437. Implants with an isotropic, i.e. one without dominating direction of the surface irregularities, an Sa value of approximately 1.5µm and an increased surface area enlargement (Sdr) of approximately 50% have shown stronger bone integration than smoother or rougher implants38.

Methods to alter microtopography Turning. The original Brånemark (Nobel Biocare) implant was a turned titanium screw with no further surface treatment37. It had a minimally rough surface and was for a long time the most used implant with good long-term clinical results39,40. Today most of the commercial implants available are minimally to moderately rough41.

Grit-blasting with various hard ceramic particles such as alumina (AlO3), titanium oxide (TiO2), silica or calcium phosphate is one way of roughening the implant surface. The size of the blasting particles determines the roughness created and the blasting particles should be chemically stable and biocompatible. Several in vivo studies have shown significantly improved bone-to-implant contact (BIC) for TiO2 blasted implants as compared to machined ones34,35,42 and clinical studies have demonstrated good results with implants in function 5 and 10 years after implantation43-47. Figure 1a shows a SEM image of a TiO2 blasted surface (TiOblast™, Astra Tech AB, Sweden). Blasting with calcium phosphates, such as hydroxyapatite and beta-tricalciumphosphates have demonstrated higher BIC values when compared to machined surfaces in some in vivo studies48,49.

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a) TiOblast™ b) Osseotite® c) TiUnite™

Figure 1. SEM images in x3000 magnification of common surface modifications.

Acid-etching of a surface with strong acids such as HCl, H2SO4, HNO3 and HF creates an isotropic surface that may enhance osseointegration in vivo50,51. The Osseotite surface (3i Biomet, USA) is produced with a mixture of HCl and H2SO4 which creates a dual-acid etched surface when heated (Figure 1b). High clinical survival rates >97%, 3 years after implantation have been reported52 for this surface. Etching with hydrofluoric (HF) acid roughens the surface and in addition changes the surface chemical composition by fluoride incorporation, which, in vivo, has been demonstrated favourable for the osseointegration8,53. The effect of surface modification with fluoride ions is suggested to be increased proliferation and differentiation of mesenchymal cells54. Osseospeed™ (Astra Tech AB, Sweden) and SLA (Straumann, Swizerland) are examples of implants manufactured by a combination of blasting and etching procedures. (Figure 2a and b shows SEM images of the two surfaces). Both implants with good clinical results reported in the literature55-57. Hypothetically the blasting procedure creates a surface roughness for good mechanical fixation and the additional etching smoothens out sharp peaks. Commonly, this combination of techniques creates a moderately rough surface.

a) b)Figure 2. SEM images in x3000 magnification of the Osseospeed™ surface (a), which is created by blasting followed by HF etching. Figure b) shows the SLA® surface modified by sand-blasting and etching with H2SO4/HCL.

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Anodization produces a micro- and or nano-porous oxide layer on the titanium surface. The appearance of the layer from the anodization process depends on current density, concentration and composition of the acids used in the electrolyte solution and the temperature. In vivo studies have demonstrated increased bone response with higher biomechanical and histological values for anodized surfaces compared to machined ones58,59. In a study by Jungner et al (2005)60, higher clinical success rates were reported for the oxidized TiUnite™ surface (Nobel Biocare, Sweden) when compared to a turned titanium implant (Mark III, Nobel Biocare, Sweden). A 5 year follow up study demonstrated good clinical results (success rate >97%) in soft bone quality despite immediate loading61. Mechanical interlocking due to bone ingrowths in the surface pores and biomechanical bonding have been suggested to be the reason for the increased performance of the anodized surface62,63. Figure 1c shows a SEM image of the moderately rough TiUnite™ surface in a x3000 magnification.

To modify the surface chemical composition, ion incorporation with magnesium, calcium or phosphorus into the oxide layer has been reported as well64. Calcium incorporation to an anodized surface increased biomechanical fixation in vivo65. A minimally rough anodized surface with calcium incorporation is manufactured by Ospol AB (Malmö, Sweden). Plasma spraying is a method where particles, hydroxyapatite (HA) or titanium are projected on the surface through a plasma torch at very high temperature. The particles condense and fuse together on the surface thereby creating a coat. Titanium plasma spraying has displayed better bone integration in vivo as compared to smoother implants66. However, clinical studies have been unable to confirm these results and have shown disadvantages with increased marginal bone resorption67,68.

Previous in vitro and in vivo studies on HA plasma sprayed titanium implants have demonstrated stronger initial bone response compared with conventional titanium implants. Further, studies comparing implants with a nanothick calcium phosphate coating and plasma sprayed calcium phosphate coated (PSCaP) implants in vivo, demonstrated significant increased early bone response and torque values to the PSCaP surface69,70.

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contact (BIC) for TiO2 blasted implants as compared to machined ones34,35,42 and clinical studies have demonstrated good results with implants in function 5 and 10 years after implantation43-47. Figure 1a shows a SEM image of a TiO2 blasted surface (TiOblast™, Astra Tech AB, Sweden). Blasting with calcium phosphates, such as hydroxyapatite and beta-tricalciumphosphates have demonstrated higher BIC values when compared to machined surfaces in some in vivo studies48,49.

a) TiOblast™ b) Osseotite® c) TiUnite™ Figure 1. SEM images in x3000 magnification of common surface modifications.

Acid-etching of a surface with strong acids such as HCl, H2SO4, HNO3 and HF creates an isotropic surface that may enhance osseointegration in vivo50,51. The Osseotite® surface (3i Biomet, USA) is produced with a mixture of HCl and H2SO4 which creates a dual-acid etched surface when heated (Figure 1b). High clinical survival rates >97%, 3 years after implantation have been reported52 for this surface. Etching with hydrofluoric (HF) acid roughens the surface and in addition changes the surface chemical composition by fluoride incorporation, which has, in vivo, been demonstrated favourable for the osseointegration8,53. The effect of surface modification with fluoride ions is suggested to be increased proliferation and differentiation of mesenchymal cells54. Osseospeed™ (Astra Tech AB, Sweden) and SLA® (Straumann, Swizerland) are examples of implants manufactured by a combination of blasting and etching procedures. (Figure 2a and b shows SEM images of the two surfaces). Both implants with good clinical results reported in the literature55-57. Hypothetically the blasting procedure creates a surface roughness for good mechanical fixation and the additional etching smoothens out sharp peaks.

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However, long-term results of plasma sprayed implants have been unfavourable and associated with failure. Plasma spraying with HA particles creates a 50-200µm thick coat but with poor adhesion to the bulk material and this is believed to be the reason for the long-term negative clinical results of such implants71. However, it was never clarified whether the positive initial bone response was due to the proposed bioactivity of HA, to possible differences in surface topography when comparing coated with non-coated implants; the Ra value for HA coated implants were at the time reported to be 1.6µm or more while a machined titanium implant is in the range of 0.5-1µm72. Furthermore, that the thicker HA-coated implants have a greater press fit when inserted in the same size defects as the controls, which may give the former a better initial stability.

Calcium phosphate coatings (CaP). Different methods are available to coat implant surfaces with calcium phosphates; for example, plasma spraying, sol-gel coating, sputter deposition, electrophoretic deposition, biomimetic precipitation etc. The Ca/P ratios may differ in these coatings, phases present may include tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate and hydroxyapatite (HA). The CaP ratio determines the degradation rate of the CaP. Pure crystalline HA is known to be the most stable and strongest among these phases. It has been demonstrated that the biological response in vitro and in vivo may differ towards differently composed CaPs; Osteoblast adhesion was evaluated towards CaP coatings with different CaP ratios (samples with CaP ratios between 0.5 and 2.5) and results demonstrated increased osteoblast adhesion with the greater CaP ratios (up to 2.5). Changes in chemistry of the different CaP ratios may have altered the osteoblast adhesion, since many proteins for osteoblast adhesion (e.g.vitronectin) have calcium binding sites which may have influenced the initial protein adsorption. Furthermore, different topography .e.g. grain and pore size) in the nanometer range between the CaP ratios may have altered the surface wettability, energy and nanometer roughness and thereby influenced the osteoblast response73. Yang et al74. compared electrochemically deposited nano-hydroxyapatite (EDHA) and biomimetically deposited CaP (BDCaP) in vivo and found signi-ficantly higher bone in contact (BIC) values and bone area for

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the EDHA after 6 and 12 weeks of healing. SEM characterisation showed that BDCaP crystals were flake like (~100nm thick and ~7μm in length), whereas the EDHA crystals were rod like with a hexagonal cross section (~70nm in diameter). The results were later confirmed by the same group75. Results as these suggest that the apatite may be advantageous over other calcium phosphates.

As mentioned previously, plasma spraying is one method to coat implants with HA, although with some disadvantages. New techniques have been developed to produce thinner CaP coatings, in the submicron range, to improve the properties and thus minimize potential problems with coat loosening.

nanoroughness and nanostructuresSurface micro topography including surface roughness of today’s available dental implants is well known, however, the characteristics of the implants on the nanometer scale of resolution are much less known. All surfaces possess nanoroughness, however not all of them have defined nanostructures. Nanostructured materials are defined in the literature as materials containing structural elements with dimensions in the range of 1 to 100nm. Furthermore, nanocoatings are defined as single or multilayer coatings with a thickness in the range of 1 to 100nm76.

Numerous in vitro studies have reported altered cell responses to differently nanostructured surfaces, however, not many studies have focused on a fundamental understanding of these observations. Therefore, the exact biological role of nanostructures is unknown. One simplified explanation may be that nanostructures attempt to mimic the biological tissues and therefore act in the same arena as the natural tissues and its extra cellular matrix.

Bone is a specialized form of connective tissue, composed by approximately 67% inorganic material, mainly hydroxyapatite (Ca10(PO4)6(OH)2); 33% organic material such as collagen (type I,V,XII), non-collagenous proteins (e.g. osteopontin, osteocalcin, phosphoproteins, bone sialoproteins, proteoglycans, glycoproteins) and water. Bone tissue may be divided into three levels of structure; nanostructures including non-collageneous proteins, collagen and hydroxyapatite. Microstructures including cells, lamella and osteons

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represent the second level. The third level including cancellous and cortical bone may be termed macrostructure. Cortical and cancellous bone is often distinguished by their degree of porosity or density, where the latter has increased porosity due to the being tissue is differently arranged with trabecula and marrow spaces77,78.

There are many ways of applying nanostructures on surfaces, for example; sol-gel coating, anodization, discrete particle deposition, ion beam deposition, alkali treatment (NaOH), lithography and printing technique. Some techniques will be described further below;

Some methods to alter nanotopographySol-gel coatings. Sols are dispersions of colloidal particles (with diameters of 1-100nm) in a liquid. A gel is an interconnected, rigid network with pores of submicrometer dimensions. During the Sol-Gel process, a liquid with a specific composition (i.e. the Sol) is converted into a solid gel phase. The particle size and aggregation is altered by the aging time, hours or days, of the Sol. Thin coatings can be deposited onto a surface by dip- or spin coating techniques. Drying removes the remaining liquid and the sample is finally heat-treated to sinter the coating. The coat thickness can be altered by the number of layers added to the surface sample. The procedure makes it possible to produce coatings of titanium, hydroxyapatite or combination of both79. In vitro studies have shown increased osteoblast adhesion and differentiation to Sol-gel coated HA titanium surfaces with a coat thickness of ≤ 1μm compared to controls80,81.

Discrete calcium deposition (DCD) makes it possible to deposit nanometer sized calcium phosphate (CaP) crystals onto a surface. This is achieved by applying a suspension of CaP crystals in the size range of 20-100nm onto the implant surface. This can be achieved using dip coating, where the implant is immersed into the suspension followed by withdrawal as a certain speed, using spin coating, where the implant is spun during or after deposition of the dispersion or by the use of spraying techniques. After the deposition step the implant is subjected to a drying procedure and optionally heat treatments. The result is a non-confluent layer of CaP crystals, with an approximately 50% coverage of the implant surface and the remaining surface being the bulk material82. The NanoTite™ (3i,

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Implant innovations, USA) is a commercially available implant with a surface produced by DAE and DCD83.

Nanocrystalline hydroxyapatite coatings may be produced by molecular self-assembly synthesis. Nanoparticles of hydroxyapatite is prepared by mixing H3PO3 and Ca(NO3) to a Ca/P ratio of 1.67 in the presence of a liquid crystalline phase. The crystalline phase limits particle growth to ~5nm. When HA particles have formed, the liquid crystalline phase is dissolved and the particles can be deposited onto a surface using DCD. The sample is dried in room temperature followed by heating in nitrogen atmosphere at 550˚C for 5 minutes to eliminate surfactants on the surface and the procedure results in a nanocrystalline HA coat (nano-HA). The thickness of the coat may be altered by adding more layers through multiple dipping of the sample84. Nanocrystalline HA may be advantageous since it resembles the nanostructure of the inorganic material in bone. Results reported by Göransson et al.85 demonstrated that surfaces coated with nano-HA, induced earlier CaP formation and in addition increased bone cell response compared to a blasted control surface in vitro. An in vivo study in rabbit tibia have demonstrated significantly increased bone formation to electro-polished cylindrical titanium implants coated with the technique described when compared to uncoated controls86.

TiO2-nanotubes are manufactured by electrochemical anodization. Oh et al.87 fabricated TiO2 nanotubes by anodization in 0.5% hydrofluoric acid solution at 20V for 30 minutes in room temperature followed by annealing at ~550˚C. This method produces anatase phase nanotubes with an outer diameter of ~100nm, inner diameter of ~80nm and a height of ~250nm. Increased osteoblast adhesion on the TiO2 nanotube surface compared to non-modified TiO2 was reported in that study87. Increased human osteoblast adhesion, proliferation and differentiation were found on a surface with TiO2 nanotube morphology compared to a polished titanium control. The nanotube surface was more hydrophilic and had increased surface roughness compared to control surface88. Significantly increased bone bonding strength to TiO2 nanotubes have beee demonstrated after 4 weeks of implantation in rabbit tibia and histological results

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whether the increased bone response is due to the surface attached fluoride ions, the different surface topography or maybe a combination of both. Figure 3a and b shows SEM images of the two surfaces, with nanostructures present on the Osseospeed™ surface.

a) Osseospeed™ c) SLActive®

b) TioBlast™ d) SLA® Figure 3a-d. Shows SEM images of the Osseospeed™(a) and SLActive® surface (c) and their predecessors TioBlast™ and SLA® in figure b) and d) respectively.

The SLActive® implant has displayed strong bone response in vivo compared to its predecessor SLA® and this was explained by SLActive® having a more hydrophilic surface. However, surface roughness differs between the two implants both on micro as well as nanometer levels of resolution41 Certainly, some techniques, such as etching, performed in an attempt to alter micro roughness may simultaneously affect roughness on the nanometer level of resolution.

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whether the increased bone response is due to the surface attached fluoride ions, the different surface topography or maybe a combination of both. Figure 3a and b shows SEM images of the two surfaces, with nanostructures present on the Osseospeed™ surface.

a) Osseospeed™ c) SLActive®

b) TioBlast™ d) SLA® Figure 3a-d. Shows SEM images of the Osseospeed™(a) and SLActive® surface (c) and their predecessors TioBlast™ and SLA® in figure b) and d) respectively.

The SLActive® implant has displayed strong bone response in vivo compared to its predecessor SLA® and this was explained by SLActive® having a more hydrophilic surface. However, surface roughness differs between the two implants both on micro as well as nanometer levels of resolution41 Certainly, some techniques, such as etching, performed in an attempt to alter micro roughness may simultaneously affect roughness on the nanometer level of resolution.

showed greater BIC values, new bone formation and calcium and phosphorus levels on the nanotube surface89. As previously mentioned, it is often difficult to change one surface characteristic, without affecting others. The Osseospeed™ surface has been demonstrated to promote the bone response compared to its predecessor TioBlast™. However, it is not known whether the increased bone response is due to the surface attached fluoride ions, the different surface topography or maybe a combination of both. Figure 3a and b shows SEM images of the two surfaces, with nanostructures present on the Osseospeed™ surface.

b) TioBlast™ d) SLA®

Figure 3a-d. Shows SEM images of the Osseospeed™(a) and SLActive® surface

(c) and their predecessors TioBlast™ and SLA® in figure b) and d) respectively.

The SLActive® implant has displayed strong bone response in vivo compared to its predecessor SLA® and this was explained by SLActive® having a more hydrophilic surface. However, surface roughness differs between the two implants both on micro as well as nanometer levels of resolution41(figure 3b and d).

a) Osseospeed™ c) SLActive®

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Certainly, some techniques, such as etching, performed in an attempt to alter micro roughness may simultaneously affect roughness on the nanometer level of resolution.

Nanostructures and biological responseWater, ions and subsequently proteins will adsorb onto the implant surface within milliseconds after insertion, the latter further interacting with cell-membrane receptors. It is suggested that nanomaterials affect protein interactions due to the unique surface properties, such as; increased surface area, surface roughness, altered electron distributions etc. Increased surface area will provide more available sites for protein adsorption and thus affect the amount of cellular interactions90. For example; Scopelliti and coworkers91 found that an increase in nanometer roughness, from 15 to 30nm, resulted in a significant increase in protein adsorption on nanostructured titanium devices. The authors suggested that proteins get trapped inside nanometer sized pores and thereby promoted protein aggregation. In vitro results reported by Liu et al.92 demonstrated enhanced osteoblast adhesion, alkaline phosphatase activity, and calcium-containing mineral deposition to nanophase titania/PLGA composites that had surface roughness values closer to that of natural bone. Webster et al.93 found enhanced osteoblast adhesion with increased nanoroughness. In the same study it was observed that greater concentrations of vitronectin was adsorbed onto the nanostructured surfaces while greater concentrations of laminin was found on the control surfaces. Unfold vitronectin may promote the availability of RGD (Arginine-Glycine-Aspartic acid) sequences that could enhance osteoblast adhesion94. Increased wettability or hydrophilicity has also been associated with increased protein adsorption and subsequently enhanced cell adhesion on biomaterials95. De Oliveira and Nanci96 found that nanotexturing of titanium-based surfaces up-regulates the early expression of bone sialoprotein (BSP) and osteopontin (OPN). The expression of BSP is generally believed to initiate newly differentiated osteoblasts that correspond with initial bone mineralization97. OPN is involved in cell adhesion, migration, and in the regulation of mineral deposition98. Webster et al.93 demonstrated significantly greater osteoblast adhesion on nanophase alumina, titanium, and hydroxyapatite

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compared to conventional formulations of the same ceramics after 4h of culturing. This study further revealed that fibroblast adhesion was significantly lower to the nanostructured surfaces which was explained by significantly increased adsorption of vitronectin on the nanostructured surfaces, while increased concentrations of laminin was found on the controls as mentioned previously. Further studies by Webster and co-workers99 demonstrated significantly greater osteoblast proliferation, more synthesis of alkaline phosphatase and deposition of calcium-containing minerals by osteoblasts cultured on nanophase ceramics compared to conventional ceramics after 21 and 28 days. Increased osteoblast adhesion and significantly more calcium and phosphorus deposition have been reported to metals with nanotopography (Ti, Ti6A14V, CoCrMo) compared to conventional metals100,101. De Oliveira et al.102 reported enhanced osteogenesis on titanium discs with chemically produced nanotopography. Increased osteoblast response on nanometer carbon fibres have been reported in the literature103,104. Sato and co-workers105 found significantly greater osteoblast adhesion on nano-titanium and nano-HA coatings compared to uncoated controls. Puckett et al.106 investigated bacteria adhesion towards different nanostructured surfaces and found significant differences in bacteria adhesion, indicating that different surfaces may be more prone to microbial colonisation and that it may be possible to influence the risk of microbial colonisation by altering the surface topography and thereby decreasing the risk of complications of peri-implant infections.

Numerous in vitro studies have shown increased cell response to different nanostructured surfaces and it is clear that nanostructures affect the cell response in different ways. Further, several in vivo studies have investigated the importance of different nanometer sized structures on different kinds of implant design in early bone healing and some have confirmed the in vitro result by demonstrating positive biological effects. Bjursten et al.89 investigated different surfaces, TiO2 gritblasted and TiO2 nanotube surfaces. Discs were implanted in rabbit tibia, and the results showed significantly improved bone bonding strength, increased bone to implant contact (BIC) and new bone formation to TiO2 nanotubes compared with the TiO2 gritblasted surface. Dual acid etched, rectangular titanium

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plates were modified with discrete crystalline deposition (DCD) of CaP nanocrystals and implanted in rat femur. Results from this study demonstrated the modified surface to have significantly higher disruption forces than non-DCD samples82. Few human studies with nanostructured surfaces have been performed. Dual acid etched (DAE) micro-implants with and without nanometer-scale CaP coating were placed in the posterior maxilla. Results from this study revealed significantly higher bone to implant contact (BIC) for the test implant compared with the control after 4 and 8 weeks of healing107. SEM was the only method for surface characterization. Similar results were reported in a study by Orsini et al.108 for the same type of implant surface. Telleman et al.83 inserted mini- implants in grafted bone with one part of the implant extended into native maxillary bone. The authors confirmed previously reported results by concluding that the CaP coated DAE surface increased the healing properties in the native bone while the difference was not seen in the bone graft area after 3 months of healing, when compared to the DAE surface alone. The surface roughness was described on the micrometer level of resolution and further with SEM. A human study investigating soft-tissue adhesion and marginal bone response, found significant increase in soft-tissue adaptation to nanoporous TiO2 after 14 weeks of healing compared to the control implant, possibly due to reduced marginal bone resorption109. These results indicate that nanostructures may also be advantageous in peri-implant soft tissue healing.

Results like these suggests that nanostructures may be clinically advantageous for shortening healing times, allowing earlier loading of dental implants and presenting increasing treatment success in patients with compromised bone.

New possibilities to evaluate cell response to nanostructuresWhether or not nanostructures, of any kind, on the implant surface are clinically relevant is not yet known, even if a few studies, previously mentioned, may suggest so. Furthermore, the mechanism behind the increased cell response to nanostructured surfaces is not fully understood. Increased understanding of the initial interaction between cells and implant surface material is of interest

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to further map the mechanism behind the varying cell and tissue response towards different surface modifications, at the micro- as well as nanometer levels of resolution. Increased understanding of the initial cell response would be of great value to further develop more optimal implant surfaces. However, there has been a lack of suitable methods to investigate and monitor real-time initial cell-surface interactions. The most commonly used in vitro technique to evaluate cell response including attachment, spreading, calcium and phosphorus deposition etc. towards different surfaces is cell culturing.

Two promising methods to in real-time evaluate implant surfaces in vitro, are optical trapping (OT) and quartz crystal micro balance with dissipation monitoring (QCM-D). The optical trap or optical tweezers was discovered in the early 1970s110,111 and has since then been used for manipulating single cells as well as investigating biophysical/biomechanical systems. The traps can be used in different applications such as transporting cells to specific locations, cell sorting, transporting foreign materials into cells as well as studying cell interactions and cell adhesion112-118. The advantages of using the optical trap include non-contact force for manipulation of cells and the possibility of measuring interaction forces in the piconewton range119. The QCM-D has become a popular tool to study bimolecular adsorption onto different surfaces. The technique enables in situ measuring without invasive labelling and is also capable of monitoring the viscoelastic properties of the adhered layer. The method has previously been used to study protein absorption and only a few studies have aimed at studying cell-surface interactions120-125.

summary and relevance of the present thesisNanostructures have been suggested to be important in the osseo-integration process since it is on the nanometer level that ions and proteins attach to the surface which may govern the following cell response and thereby the early bone healing around the implant. However, our knowledge of the exact mechanisms behind demonstrated in vitro and in vivo results and to what extent nano-structures influence the early bone healing and subsequently the osseointegration process is still very limited. Two novel promising

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methods were evaluated in this thesis to investigate the possibility to detect differences in the initial cell adhesion towards different surfaces.

There are a number of ways to alter implant surface properties, such as chemistry and topography, but as mentioned, it is difficult to change one without affecting the other. Hence, it is hard to evaluate and interpret from what precise characteristics the altered biological response is influenced. To evaluate the effect of nanostructures in absence of micro topography, cylindrical implants with applied nanostructures with different properties were investigated in this thesis.

Processes aiming at modifying the surface at the micrometer level, will most certainly affect the topography at the nanometer level. Therefore, to fully understand the relationship between the properties of an implant surface and its osseointegration behaviour, a characterization at the nanometer level of resolution is relevant. The first study in this thesis aimed at characterizing nanometer topography on screw-shaped dental implants, with different surface modifications.

Calcium phosphate coatings have shown increased biological responses and some studies have suggested that the hydroxyapatite may be more advantageous than other calcium phosphates. Blasted and acid etched screw-shaped implants have previously demonstrated good biocompatibility and clinical results with high success rates. The possibility of nanometer coatings to further promote bone healing when applied on a blasted and etched screw-shaped implant has not been investigated. Therefore, in vivo studies in this thesis aimed at investigating if additional nanostructures on micrometer rough implants may improve the biological response in bone of different qualities.

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aims

Surface characterization;• To describe a technique to characterize commercial oral

implants on the nanometer level and to investigate whether or not the nanometer surface roughness was correlated to the more well-known micrometer roughness on the implants.

Real-time in vitro;• To study the real-time initial cellular interactions of human

osteoblasts and fibroblasts to different implant surfaces with and without a coat of nanocrystalline hydroxyapatite using optical trapping and QCM-D.

In vivo bone response;• To evaluate the early bone response to a nanocrystalline

hydroxyapatite coating compared to a nano titanium coating when deposited on cylindrical titanium implants without microstructures.

• To investigate whether nanometer thick coatings of hydroxyapatite nanocrystals applied on moderately rough surfaces may enhance early bone healing on screw-shaped implants and to evaluate if the thickness of the coat influences healing.

• To investigate whether a sub-micron coating of hydroxyapatite nanocrystals applied on a moderately rough surface may enhance early bone healing on screw-shaped implants in trabecular bone.

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materials anD methODs

implants and componentsImplant design and surface preparationIn study I, commercial dental implants of different brands with 12 different surface modifications were used. Rectangular cubes were manufactured from commercially pure (grade 4) titanium (Ti) for study II. Four samples of each surface and four different surface modifications were used. 1) Oxidized Ti, 2) Oxidized Ti coated with nanocrystalline HA (nano-HA), 3) Turned Ti and 4) Turned Ti coated with nano-HA. The nano-HA coating was performed using a technique described by Kjellin et al84. Cylindrical implants sized 8 mm in length and 3.5 mm in diameter manufactured from commercially pure titanium (grade 3) were used in study III. Each implant had an upper threaded part fixed in a plate to stabilize the implant. The whole implant device was fixed in the bone through two side holes for fixating screws. 20 implants were divided in two groups; one group was coated with nano-HA. The other group was coated with nano-titania (provided by MetAlviveTM, Vivoxid, Turku, Finland). For study IV and V screw shaped implants with a length of 8.5 mm and a diameter of 3.5 mm were manufactured from commercially pure titanium (grade 4). The surfaces were blasted with hydroxyapatite particles 6.5-9µm in size and then acid etched. The test surfaces in study IV were coated with two different thicknesses of nanocrystalline hydroxyapatite, deposited as described by Kjellin at al84 and in study V a modified technique was used, which resulted in a thicker submicron coating of nanosized hydroxyapatite crystals.

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Paper: I II III IV V

12 commercially available dental implants with different surface modifications.

Turned Ti

Turned Ti + nano-HA

Oxidized Ti

Oxidized Ti + nano-HA

Cylindrical implant + nano-HA

Cylindrical implant + nano-titanium

Sandblasted/Acid etched surface

Sandblasted/Acid etched + nano-HA single coat

Sandblasted/Acid etched + nano-HA double coat

Sandblasted/Acid etched + submicron thick nano-HA coat Table 1. Titanium surface modifications used in the studies in this thesis.

methods for surface characterizationAtomic Force Microscope With an Atomic force microscope (AFM) it is possible to characterize surfaces near the atomic level and therefore suitable to evaluate nanometer structures. The AFM consists of a cantilever with a sharp tip (probe) at its end that is used to scan the surface. The cantilever is typically silicon or silicon nitride with a tip radius of curvature on the order of nanometers. When the probe is close to the surface, forces between the probe and the surface lead to a deflection of the cantilever according to Hooke›s law. The AFM can be operated in several different modes, contact and non-contact modes, depending on the application. In a tapping mode, AFM images are produced by imaging the force of the intermittent contacts of the probe with the surface. The cantilever is driven to oscillate near its resonance frequency and the amplitude of this oscillation is approximately 100 to 200nm. Due to the interaction of forces acting on the cantilever when the probe comes close to the surface for example, Van der Waals force, dipole-dipole interaction and electrostatic forces, cause the amplitude of this oscillation to decrease as the probe gets closer to the surface. The tapping mode reduces the damage done to the surface and the probe compared to contact mode. The maximal lateral measuring area is 100x100µm and resolution about 100pm. Maximal vertical distance

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is 6µm and the level of resolution close to 10pm. The AFM analysis in study III, were performed using Dimension 3000 SPMTM instrument (Digital instruments, Santa Barbara, USA).

Optical interferometryThe principal of optical interferometry is based on the principles of interference. A light beam (λ: 550nm) is divided and one beam is directed towards the sample surface and the other towards a reference plane. Irregularities at the sample surface will affect the reflected light and cause phase changes; this will either augment or cancel out the reference waves. The surface roughness was examined using a white light interferometer (MicroXAMTM Phaseshift, Arizona, USA). The maximal resolution of the technique is 0.3μm horizontally and 0.05nm vertically. In these studies an x50 objective and a zoom factor of 0.62 was used. Each area measured had a size of 264x200μm. With this instrument it is possible to evaluate surface roughness at the micrometer level of resolution in height and spatial directions. To describe the surface roughness on the nanometer level of resolution, it is possible only in terms of height deviation. Horizontally, the wavelength of light is the limiting factor.

Gaussian filter and surface parametersTo describe the surface topography, the surface roughness, the waviness, and shape of the implant must be taken into consideration. The standard filter used to separate roughness from waviness and shape for digital three-dimensional measurements on a micrometer level is a Gaussian filter. A high-pass filter significantly reduces the impact of structures larger than the filter size used. A low-pass filter can also be applied to evaluate how much of the shape, waviness and roughness that were removed by the high-pass filter (Figure 4.).

a) Original image b) High pass filter c) Low pass filterFigure 4. Surface roughness on a micrometer level for a blasted implant surface. A) Original image without any filter applied. B) Image after filtering process using a 50x50µm high pass gaussian filter. C) Image after applied 50x50µm low pass filter.

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a) Original image b) High pass filter c) Low pass filter Figure 4. Surface roughness on a micrometer level for a blasted implant surface. A) Original image without any filter applied. B) Image after filtering process using a 50x50µm high pass gaussian filter. C) Image after applied 50x50µm low pass filter.

A high-pass Gaussian filter size of 50x50µm have previously been suggested to be suitable for evaluating threaded implants on the micrometer level of resolution126. To be able to evaluate the nanometer roughness, the micrometer roughness must be considered as waviness and a suitable filter size to eliminate it must be used. Surfascan software (Somicronic Instrument, Lyon, France) was used to do the filtration and evaluation of the interferometry and AFM measurements. This software provides numerical descriptions and images of the surface topography. Images of the surfaces were also obtained using SPIPTM software (Scanning Probe Image Processor, Image Metrology, Denmark). For numerical description of the surface topography there are hundreds of parameters available. R-parameters describe the surface topography in two-dimensions, while S-parameters are three dimensional. The Sa value is most frequently used when describing surface topography and implants with a Sa ~1.5 µm and an isotropic surface, i.e. a surface with no dominating direction of the irregularities have shown stronger bone integration than smoother and rougher implants127. Mathematical formulas for the parameters can be found in the literature128. The following parameters were used in this thesis: Sa (µm) = The amplitude parameter describes the arithmetic mean of the surface roughness from a mean plane.

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A high-pass Gaussian filter size of 50x50µm have previously been suggested to be suitable for evaluating threaded implants on the micrometer level of resolution126. To be able to evaluate the nanometer roughness, the micrometer roughness must be considered as waviness and a suitable filter size to eliminate it must be used. Surfascan software (Somicronic Instrument, Lyon, France) was used to do the filtration and evaluation of the interferometry measurements. This software provides numerical descriptions and images of the surface topography. Images of the surfaces were also obtained using SPIPTM software (Scanning Probe Image Processor, Image Metrology, Denmark).

For numerical description of the surface topography there are hundreds of parameters available. R-parameters describe the sur-face topography in two-dimensions, while S-parameters are three dimensional. The Sa value is most frequently used when describing surface topography and implants with a Sa ~1.5 µm and an iso-tropic surface, i.e. a surface with no dominating direction of the irregularities, have shown stronger bone integration than smoother and rougher implants127. Mathematical formulas for the parame-ters can be found in the literature128. The following parameters were used in this thesis:

Sa (µm) = The amplitude parameter describes the arithmetic mean of the surface roughness from a mean plane.

Sds (µm-2) = This spatial parameter describes the density of summits, i.e. number of peaks per area unit.

Sdr (%) = A hybrid parameter that describes the ratio between the developed surface area added by the roughness and a flat reference area.

Sci = Core fluid retention index. This functional parameter is related to the volume that the surface is able to contain. Based on earlier studies on a micrometer level of resolution, a low value might be related to a positive biological outcome of bone anchored implants129.

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Scanning Electron MicroscopyScanning Electron Microscopy (SEM) was used for characterization of the surface topography on the micrometer and nanometer level. SEM analysis was performed on the implant surfaces using a Leo Ultra 55 FEG high resolution scanning electron microscope (Carl Zeiss SMT Inc., North America), operating at an acceleration voltage of 5-10 kV. The micrographs were recorded at randomly chosen areas of the implants using the secondary electrons.

X-ray Photoelectron Spectroscopy The surface chemical compositions can be analysed with X-ray Photoelectron Spectroscopy (XPS). The measuring depth is approximately 10nm, however dependent on the material analysed, hence information of the sample surface is provided. The method involves X-ray irradiation from a mono-energetic source onto a solid sample, which is performed under ultra-high vacuum. Upon absorption electrons are ejected from the sample and their kinetic energy are analysed. Each element is associated with a certain binding energy and will therefore give a characteristic set of peaks and a unique spectrum. Qualitative analysis can be performed since the kinetic energy of a photoelectron is uniquely dependent on the element and its chemical state. The XPS survey spectra in these studies were obtained using a PHI 5000C ESCA System (Perkin–Elmer Wellesley, USA). A α excitation source was used at 250 W with an operating angle of 45°.

Powder X-ray Diffraction (PXRD)The technique is used for identification of the crystal structure of a material. The electron cloud of atoms scatters X-rays and if there are enough atoms the scattering can interfere. If the interference is constructive and coherent (the scattering waves are in phase) diffracted X-rays in a specific direction is obtained. Crystals diffract X-rays since they are periodic with a periodicity in the same length scale as X-rays. With the use of Bragg’s law which relates the wavelength of the beam to the distance between parallel planes of ordered species, i.e. atoms in a crystal, it is possible to distinguish between different structures of crystalline powders. XRD was performed using a Bruker XRD D8 Advance (Bruker AXS, Karlsruhe, Germany) and monochromatic Cu radiation.

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methods for evaluation in vitroCells and cultureHuman osteoblasts and human gingival fibroblasts were used for the experiments in study II. All cells were maintained in a humidified incubator (5% CO2/95% Air atmosphere at 37°C).

Human mandibular bone specimens without any clinical or radiographic evidence of pathology and human gingival fibroblasts were obtained from biopsies taken during surgery at the department of Oral and Maxillofacial Surgery, University of Bergen, Norway. All donors were females 18-38 years old, with no known systemic disease.

Fibroblasts; The cells were grown in DMEM culture medium with low glucose (1g/l), L-Glutamine, supplemented with 10% featal bovine serum (FBS) and 1% Penicillin/Streptomycin. At 90% confluence, the cells were detached from the substrate by treatment with trypsin-EDTA (PAA Laboratories, Fisher Scientific) for 5 min in an incubator at 37°C, centrifuged for 5 min at 1000 rpm at 20°C and re-suspended in complete culture medium before transferred to the QCM-D instrument at a concentration of ~750 000 cells /ml. Cell numbers were determined by direct counting and the final cell suspension was kept in a centrifuge tube at 37°C before injected into the QCM-D instrument. The same procedure was done before transferring the cells to the optical trap, however at a much lower cell concentration.

Osteoblasts; The osteoblasts were cultured in MEM, alpha modification with Ribonucleosides and Deoxyribonucleosides (Gibco, Invitrogen), supplemented with 1% penicillin/streptomycin solution and 10% FBS (PAA Laboratories, Fisher Scientific). The cells were detached from the substrate by the same protocol as the fibroblasts and were then transferred to the QCM-D instrument at a concentration of 1x106/ml. The same procedure was done before transferring the cells to the optical trap, however, at a much lower cell concentration.

Optical trappingThe optical trap induces forces exerted by a strongly focused laser beam. The trap can be described as being composed of two independent forces. One is the gradient force that is created in the

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direction of the field gradient as a result of fluctuating dipoles that are formed in the trap’s inhomogeneous electric field. This force depends on the dielectric properties of the gradient intensity and the trapped object. The other force, the scattering force that is due to the momentum transfer from the photons to the trapped object, i.e., the object is pushed in the same direction as the incoming beam. The gradient force must exceed the scattering force to create a stable trapping of the object. This can only be achieved if the beam diverges rapidly enough which is ideally performed using microscope objective lenses in combination with computer controlled microscopes130,131. The optical trap system for study II was designed as follows; a mmi Cell Manipulator was set up on a standard Nikon Eclipse TE 2000-U inverted scientific microscope with x60 Plan Apo objective (Nikon instruments). The optical trap laser used was a 3W Nd:YAG (yttrium aluminium garnet) laser emitting light at a wavelength of 1064 nm. A galvanometer, based on optical scanners, which could create up to 11 individual traps simultaneously using at time sharing mode was used in the set-up. To measure the position of the trapped cell, a position detector module containing a four segment photodiode quadrant detector (QD) was used. A high resolution digital three chip camera charge coupled device (CCD) used for observation was coupled to the system.

The cell attachment measurements were performed as follows for both osteoblasts and fibroblasts; two surface samples were attached towards each other with a small space between them to a cover slip. Cell suspension was then put between the surface samples. A single cell was trapped in the suspension and had its size measured, then moved towards one of the surfaces and held in direct contact with the surface to promote adhesion. The cell was then attempted to be removed from the surface with the trap and if successful, the cell was moved against the opposite surface and the adhesion procedure was repeated. In a previous study results showed that, in case of cell attachment to the surface, the cell adhered with a force so strong that it could not be removed with the trap130. The experiments were repeated both ways to not give one surface advantage.

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Quartz Crystal Microbalance with dissipation monitoring (QCM-D)The QCM-D instrument utilizes a piezoelectric quartz crystal sensor, which has metal electrodes on each side, most often gold. When an AC-voltage is applied over the electrodes, the crystal can be made to oscillate at its resonant frequency. The principle of QCM-D is that the frequency of the crystal changes when the mass of the crystal changes, for example if a protein or a cell attach onto the surface. The resonance frequency, decreases when the mass attached to the surface increases and the frequency can be measured with a very high precision and correspond to very small amounts of mass. When a flexible mass, like a cell attaches to the surface non-rigidly and deforms during oscillation, it causes dissipative energy losses. The dissipation factor (D) is the sum of all energy losses in the oscillating system and it gives information about the interfacial coupling of the attached cell to the crystal surface. In study II, a QCM-D (Q-Sense E4 from Q-sense AB, Gothenburg, Sweden) was used. This method measures the resonant frequency (f) and the dissipative energy (D) of the oscillating system on four sensors simultaneously to enable four parallel measurements (Ismatec SA, Glattbrugg, Switzerland). All the experiments were performed at a temperature of 37°C. At the end of the experiment, the cells attached to the crystals were fixed in 3.7% formaldehyde in PBS and extracted in 0.1% triton X-100 in PBS. Further, stained with Alexa Fluor®555 phalloidin, to enable fluorescence microscopy. Images of the cells were acquired with a microscope together with a camera (Axioplan 2 and Axiocam, Carl Zeiss AB, Stockholm). A light microscope (Olympus BH-2) and a camera (Olympus DP12) were used in order to calculate the cell surface coverage on each crystal.

methods for evaluation in vivo Animals and surgical techniqueThe animal studies were approved by the Gothenburg ethical committee.

10 male New Zealand White rabbits were used in study III. One test and one control implant device were placed in each animal. For study IV and V, 27 adult female New Zeeland White rabbits were used. Each rabbit received six implants, two in each proximal

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tibia and one in each femoral metaphysis and therefore each animal served as its own control. All operations were performed under aseptic conditions. The implant sites were drilled under saline irrigation and the implants were inserted by one operator in a randomized manner. Immediately after surgery the rabbits were kept in separate cages to control the wound healing. They had free access to tap water and were fed with pellets and hey. After initial healing the rabbits were kept so that they could run free in a specially designed room. After complete healing time, the animals were sacrificed with an overdose of pentobarbital 60mg/ml (Pentobarbitalnatrium, Apoteksbolaget, Sweden).

Removal torque analysisBy using a specially constructed, electrically controlled removal torque unit, it is possible to evaluate the torque needed to interrupt osseointegration of an implant. The electrically controlled removal torque unit enables precise readings of torque figures that may be recalculated into shear forces. The implants were subjected to the torque (Ncm), only to interrupt osseointegration but were then not screwed out from the bone any further in order to have the sample as intact as possible for the histological evaluation.

Histological specimen preparation and evaluationEach implant was removed in a block with the surrounding bone tissue and fixed in 4% neutral buffered formaldehyde. The specimens were then dehydrated in series of ethanol solutions and embedded in light curing resin (Technovit 7200 VLC, Kultzer & co, Germany). Un-decalcified sections were then cut and ground as described by Donath132. The final sections were approximately 15-20µm thick and stained with toluidine-blue. The histological evaluations were performed using a light microscope and image analysis software (Image analysis 2000, Sweden). Qualitative and quantitative analysis were performed including; bone in contact (BIC), bone area (BA) and amount of newly formed bone (NB). All measurements were performed in a blinded manner.

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Data analysis/statisticsStatistical analysis was performed using SPSS (statistical package for the social studies). In study I the implants were evaluated for significant differences in surface topography on both micro- and nanometer level of resolution. The data were analysed with one-way ANOVA with the significance level set at p≤0.01.

In study III the histological evaluations were performed with Wilcoxon sign rank test with considered significance at p≤ 0.05. The interferometer surface characterization in study IV was analysed using one-way ANOVA. Differences were considered significant at p≤0.05. Torque results, bone area and newly formed bone were evaluated using Kruskal-Wallis and also with Analysis of Variance (ANOVA). The assumption of the ANOVA was tested with Levene’s test of equal variances, and if needed the data were log10 transformed. Differences were considered significant at p≤0.05. In study V, Mann-Whitneys U-test was used for all evaluation and differences were considered significant at p≤ 0.05.

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results

surface characterisationSEMFigure 5 shows SEM images in x3000 magnification on different implant surfaces modifications in study I. The images show a homogenous honeycomb-like structure for the etched implants, SLA and the 3i implants. The blasted implants, TioBlast, Osseospeed, Southern implant, Lifecore RBM and Dentatus blasted, had a similar mountain-like structure. The turned surfaces all gave similar SEM images. Different surface topography was seen, at a magni-fication of x10.000, on the Osseospeed surface compared to the other blasted implants. A honeycomb-like structure, similar to the one found on etched implants, was found partly on the Osseospeed implant (figure 3a). The TiUnite implant had the most unique surface among those investigated. At x3000 magnification, the surface resembles volcano-like structures (figure 5d).

Figure 5. SEM images of an etched (a), blasted (b), turned (c) and anodized (d) surface.

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RESULTS

Surface characterisation SEM Figure 5 shows SEM images in x3000 magnification on different implant surfaces modifications in study I. The images show a homogenous honeycomb-like structure for the etched implants, SLA and the 3i implants. The blasted implants, TioBlast, Osseospeed, Southern implant, Lifecore RBM and Dentatus blasted, had a similar mountain-like structure. The turned surfaces all gave similar SEM images. Different surface topography was seen, at a magni-

fication of x10.000, on the Osseospeed surface compared to the other blasted implants. A honeycomb-like structure, similar to the one found on etched implants, was found partly on the Osseospeed implant (figure 3a). The TiUnite implant had the most unique surface among those investigated. At x3000 magnification, the surface resembles volcano-like structures (fig.5).

a

b

c

d

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In x100.000 magnifications, structures in the nanometer range are visible on the TiUnite as well as the 3i Nanotite surface (Figure 7).

SEM images from study IV and V of the sand blasted and acid etched implants with and without nano-HA coating are shown in figure 6. Nanosized structures could be seen on both coated and un-coated surfaces.

a) b) c) d) Figure 6. SEM images of the implants used in study IV and V. Sandblasted and acid etched controlsurface (a), with an applied single coat of nano-HA (b), double coated nano-HA (c) and a submicron thick nano-HA coat (d).

Interferometry characterizationResults from study I showed that a 1x1µm Gaussian filter was useful for identifying nm roughness with respect to height deviation. The commercial dental implants did present nanometer roughness of various amounts. The mean surface roughness (Sa) on the nanometer level ranged between 0.012-0.073µm and significant differences were found between the implants. The nanometer roughness did not correspond with surface roughness on the micrometer level of resolution when comparing Sa values. Images of the surface roughness on the nanometer level are shown in figure 7. The low-pass filter indicates how much shape, waviness and microroughness that have been removed with the Gaussian high-pass filter.

The numerical description of the surface topography for the surfaces in study IV and V are presented in table 2. No significant differences in Sa values were found on the micro- or nanometerlevel of resolution.

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In x100.000 magnifications, structures in the nanometer range are visible on the TiUnite as well as the 3i Nanotite surface (Figure 7). SEM images from study IV and V of the sand blasted and acid etched implants with and without nano-HA coating are shown in figure 6. Nanosized structures could be seen on both coated and un-coated surfaces.

a) b) c) d) Figure 6. SEM images of the implants used in study IV and V. Sandblasted and acid etched controlsurface (a), with an applied single coat of nano-HA (b), double coated nano-HA (c) and a submicron thick nano-HA coat.

Interferometry characterization Results from study I showed that a 1 x 1µm Gaussian filter was useful for identifying nm roughness with respect to height deviation. The commercial dental implants did present nanometer roughness of various amounts. The mean surface roughness (Sa) ranged between 0.012-0.073µm and significant differences were found between the implants. The nanometer roughness did not correspond with surface roughness on the micrometer level of resolution when comparing Sa values. Images of the surface roughness on the nanometer level are shown in figure 7. The low-pass filter indicates how much shape, waviness and microroughness that have been removed with the Gaussian high-pass filter. The numerical description of the surface topography for the surfaces in study IV and V are presented in table 2. No significant differences in Sa values were found on the micro- or nanometerlevel of resolution.

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Gausfilter 50x50µm Gausfilter 1x1 µm

Sa(µm) Sds(/mm2) Sdr(%) Sci Sa(nm) Sds(/mm2) Sdr(%) Sci

-Control1,08

(0.41)1 184807 142.5 1.21

114(11.1)

2 055650 74.9 0.95

-Single coat nano-HA

0.91(0.20)

1 222269 117.4 1.23111(7.9)

2 068575 73.1 0.86

-Double coat nano-HA

0.77(0.19)

1 282006 119.3 1.43118(6.2)

2 176006 84.1 0.79

-Submicron nano-HA

0.93 (0.25)

1 259841 146.5 1.12119 (6.9)

2 132025 83.9 0.84

Table 2. Results from interferometer characterization in study IV and V. The numbers represent the mean value of each parameter. (Standard deviation for the Sa is presented in parentheses).

a) Osseospeed™ b) NanoTite™ c) TiUnite™Figure 7. The upper images show SEM images in x100.000 magnification. The middle row shows interferometry measurement with an applied 1x1µm high pass Gaussian filter. The lower row shows the surfaces with an applied 1x1µm low pass Gaussian filter.

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Table 2. Results from interferometer characterization in study IV and V. The numbers represent the mean value of each parameter. (Standard deviation for the Sa is presented in parentheses).

a) Osseospeed™ b) NanoTite™ c) TiUnite™ Figure 7. The upper images show SEM images in x100.000 magnification. The middle row shows interferometry measurement with an applied 1x1µm high pass Gaussian filter. The lower row shows the surfaces with an applied 1x1µm low pass Gaussian filter.

Gausfilter 50x50µm Gausfilter 1x1 µm

Sa(µm) Sds(/mm2) Sdr(%) Sci Sa(nm) Sds(/mm2) Sdr(%) Sci

-Control 1,08 (0.41)

1 184807 142.5 1.21 114 (11.1)

2 055650 74.9 0.95

-Single coat nano-HA

0.91 (0.20)

1 222269 117.4 1.23 111 (7.9)

2 068575 73.1 0.86

-Double coat nano-HA

0.77 (0.19) 1 282006 119.3 1.43

118 (6.2) 2 176006 84.1 0.79

-Submicron nano-HA

0.93 (0.25) 1 259841 146.5 1.12

119 (6.9)

2 132025 83.9 0.84

48 62

AFM AFM measurements in study III revealed higher Sa and Sdr values on the nano-HA surface when compared to nano-titania on the lateral walls of the cylinder. However, at the apical part of the cylinder (not polished prior coating) the nano-titania presented higher Sa and Sdr values compared to the nano-HA. XPS and PXRD In study IV and V, the XPS analyses showed presence of calcium and phosphorus on the nano-HA coated test implants while the controls had no such elements present (Figure 8). Furthermore, the XRD demonstrated the presence of crystalline HA.

a) Nano-HA coated implant surface.

b) Un-coated implant surface. Figure 8. a) XPS spectra showing the presence of calcium and phosphor on the nano-HA surface and b) XPS spectra on the control surface verifying the absence of such elements.

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AFM AFM measurements in study III revealed higher Sa and Sdr values on the nano-HA surface when compared to nano-titania on the lateral walls of the cylinder. However, at the apical part of the cylinder (not polished prior coating) the nano-titania presented higher Sa and Sdr values compared to the nano-HA. XPS and PXRD In study IV and V, the XPS analyses showed presence of calcium and phosphorus on the nano-HA coated test implants while the controls had no such elements present (Figure 8). Furthermore, the XRD demonstrated the presence of crystalline HA.

a) Nano-HA coated implant surface.

b) Un-coated implant surface. Figure 8. a) XPS spectra showing the presence of calcium and phosphor on the nano-HA surface and b) XPS spectra on the control surface verifying the absence of such elements.

AFMAFM measurements in study III revealed higher Sa and Sdr values on the nano-HA surface when compared to nano-titania on the lateral walls of the cylinder. However, at the apical part of the cylinder (not polished prior coating) the nano-titania presented higher Sa and Sdr values compared to the nano-HA.

XPS and PXRDIn study IV and V, the XPS analyses showed presence of calcium and phosphorus on the nano-HA coated test implants while the controls had no such elements present (Figure 8). Furthermore, the XRD demonstrated the presence of crystalline HA.

a) Nano-HA coated implant surface.

b) Un-coated implant surface.

Figure 8. a) XPS spectra showing the presence of calcium and phosphorous on the nano-HA surface and b) XPS spectra on the control surface verifying the absence of such elements.

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Evaluation in vitroOptical trapResults from the optical trap experiment for the osteoblasts and fibroblasts in study II are presented in figure 9.

Figure 9. Mean percentages of all attached osteoblasts (left) and fibroblasts (right) onto the different surfaces. Both cell types showed similar response when comparing turned versus turned+nano-HA and oxidized versus oxidized+nano-HA. Results from turned versus oxidized surfaces differed when comparing osteoblast and fibroblast response.

When comparing the turned surface versus the turned+nano-HA, the turned presented more attached osteoblasts. The oxidized+nano-HA surface showed higher percentage of attached cells when compared to the oxidized only. When comparing the turned versus the oxidized, the turned presented the higher value. As for the osteoblasts, the turned surface presented more attached fibroblasts when compared to turned+nano-HA. Furthermore, the oxidized+nano-HA surface had higher values when compared to the oxidized. In contrast to the osteoblasts, the fibroblasts tested on the oxidized surface had higher percentage of attached cells when compared to the turned surface. QCM-DVarying magnitude of dissipation was seen for the different surfaces for both osteoblasts and fibroblasts measurements. An initial decrease in frequency was observed followed by an increase and thereafter the frequency was fluctuating and in general small decreases in frequency was observed. The frequency decreased more for the osteoblast measurements compared to the fibroblast. The dissipation and frequency for the osteoblast and fibroblast measurement are presented in figure 10.

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Figure 10. Recorded dissipation (A) and frequency shifts (B) over time for the adhesion of osteoblasts onto the titanium (red and blue) and nano-HA(black). Dissipation (C) and frequency (D) shifts recorded for the fibroblasts onto titanium (red, green, pink) and nano-HA (black, blue, yellow) crystals. Different degree of surface coverage was reached which is seen by the varying magnitude of dissipation (A,C).

The final shift in dissipation for each fibroblast and osteoblast measurement were plotted against cell surface coverage (CSC) and is presented in figure 11 a-b; showing that dissipation increased with increasing percentage surface coverage. No such correlation was found when frequency was plotted against CSC for either of the two cell types (figure 11 c-d).

In figure 12, the osteoblast and fibroblast spreading was compared (left graph). The osteoblasts had a tendency for delayed spreading on the surface compared to the fibroblasts. No difference in cell behavior upon initial adhesion onto the different surfaces could be detected when comparing the recorded shifts in dissipation. (Figure 12, right graph)

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a) b)

c) d)

Figure 11. The final shift in dissipation after the adhesion of fibroblasts (a) and osteoblasts (b) onto nano-HA and Ti QCM-crystals plotted against final surface coverage (CSC) percentage. The final shift for frequency plotted against CSC for the fibroblasts (c) and osteoblasts (d).

Figure 12. The left graph shows the dissipation shift for the adhesion of fibroblasts (blue) and osteoblasts (black). Right graph: Dissipation shifts for osteoblasts and fibroblasts on titanium (green) and nano-HA (red).

Figure 13 presents images from the fluorescence microscopy and show spreading of osteoblasts and fibroblasts on titanium and nano-HA surfaces.

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a) b) c) d)

Figure 13. Osteoblast adhesion on a titanium QCM-D crystal (a) and to a nano-HA coated crystal (b). Fibroblast adhesion on a titanium QCM-D crystal (c) and to a nano-HA coated crystal (d).

evaluation in vivoRemoval torque analysisThe removal torque analyses from study IV and V did not show any significant differences between the test and control implants at any healing time. In study IV the double coated nano-HA showed a tendency for a higher removal torque after two weeks of healing, the same was noted for the nano-HA coated implant in study V.

Histological evaluationQualitative analyses of all the samples from study III-V showed a normal inflammatory response in terms of few macrophages and neutrophiles observed in the histological samples. BIC was evaluated in study III, there were no significant differences between the nano-titania and the nano-HA when measured on the lateral walls of the implants. However, the nano-titania showed significantly (p≤0.05) higher BIC values in the apical region. In study IV and V there were no significant differences in the amount of newly formed bone after 2, 4 or 9 weeks of healing between the differently modified surfaces. However, in study IV there was a tendency for more new bone formation on the single nano-HA coated implant compared to the control. When evaluating the bone area, it was found that it increased with time for all the implant surfaces in study IV and V. No significant differences were noted in study III or IV. The three best threads on each side of the histological samples were evaluated in study V, the control implant had significantly (p=0.025) higher values than the nano-HA after 9 weeks of healing. Furthermore, when evaluating the BA on the upper non threaded part of the implants there was a significantly (p=0.003) higher value for the control implant.

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Images from the fluorescence microscopy show spreading of osteoblasts and fibroblasts on titanium and nano-HA surfaces are presented in figure 13.

a) b) c) d) Figure 13. Osteoblast adhesion on a titanium QCM-D crystal (a) and to a nano-HA coated crystal (b). Fibroblast adhesion on a titanium QCM-D crystal (c) and to a nano-HA coated crystal (d).

Evaluation in vivo Removal torque analysis The removal torque analyses from studiy IV and V did not show any significant differences between the test and control implants at any healing time. In study IV the double coated nano-HA showed a tendency for a higher removal torque after two weeks of healing, the same was noted for the nano-HA coated implant in study V. Histological evaluation Qualitative analyses of all the samples from study III-V showed a normal inflammatory response in terms of few macrophages and neutrophiles observed in the histological samples. BIC was evaluated in study III, there were no significant differences between the nano-titania and the nano-HA when measured on the lateral walls of the implants. However, the nano-titania showed significantly (p≤0.05) higher BIC values in the apical region. In study IV and V there were no significant differences in the amount of newly formed bone after 2, 4 or 9 weeks of healing between the differently modified surfaces. However, in study IV there was a tendency for more new bone formation on the single nano-HA coated implant compared to the control. When evaluating the bone area, it was found that it increased with time for all the implant surfaces in study IV and V. No significant differences were noted in

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DiscussiOn

implant material, design and surface characteristicsC.p. titanium is a well investigated implant material, which has demonstrated excellent biocompatibility. It is currently the most commonly used material for oral implants. C.p. titanium was used in in vitro and in vivo studies in the present thesis to verify that the biological response mainly depends on implant surface characteristics. Smooth cylindrical implants were used in study III with the aim to evaluate the nanotopography and to ensure that the bone response was not due to macro or micrometer topography alone. The model with a stabilization plate has been tested previously and proven suitable for in vivo evaluations86,133,134. In studies IV and V, screw-shaped implants were used to evaluate if applied nanostructures to an already well-known microroughness used on commercially available oral implants further may improve the early bone healing and osseointegration.

evaluation of surface characteristicsAFMAFM is suitable to evaluate nanotopography on cylindrical implants and was used in study III. The technique was not suitable for study I, IV or V since it has a limited maximum vertical measuring range of 6 µm, too narrow to evaluate many implant surfaces with modified topography. Furthermore, the threaded part of a dental implant cannot be measured with this technique which makes it unsuitable to evaluate many commercially available screw-shaped implants126.

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SEM and XPSSEM is widely used in the literature to characterize surface morpho-logy at the micro- but also on the nanometer level of resolution. XPS is a frequently used technique to describe surface chemistry. To as far as possible characterize the surface of the implants used in study IV and V, XPS and SEM were used together with optical interferometry characterisation which presented more surface details than available in paper III, where these methods were not applied.

Optical interferometryAn optical interferometer was used for evaluation of the surface roughness, on the micro- as well as nanometer level of resolution in this thesis. This method, applied together with an appropriate filtering process, is suitable for evaluating surface micro roughness on screw-shaped dental implants. Surfaces with low reflective capacity and structures with slopes over three degrees are difficult to measure and may result in invalid data126.

Gaussian filter and surface parametersFor evaluating the micrometer roughness on the screw-shaped implants, a Gaussian filter of 50x50µm was used, suitable according to Wennerberg and Albrektsson126. Due to difficulties in eliminating shape, waviness and microroughness on tops and valleys down to the level of implant nanoroughness, these analyses were limited to the flanks.

For evaluation of surface roughness one height, one spatial and one hybrid parameter was used as recommended by Wennerberg and Albrektsson126, in addition one functional parameter was analyzed. However, the recommendation regards evaluation of roughness on the micrometer level of resolution. There are hundreds of parameters available and there are others than the ones used in this thesis that may be of importance and maybe even more relevant when evaluating nanometer roughness. However, so far the Sa and Sdr value are the only parameters that have been correlated to the biological response in published animal tests.

Since several in vitro and some in vivo studies have demonstrated that nanostructures may be relevant to the osseointegration process,

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it was of great interest to explore how the surface roughness of commercial implants will appear on a nanometer level of resolution. In study I, interferometry was used to evaluate micrometer roughness and, if possible, to characterize oral implants on the nanometer level of resolution.

Results demonstrated that commercial implants do have distinct roughness at the nanometer level of resolution if in varying amounts dependent on the precise manufacturing technique of the respective implant surfaces. Significant differences in mean surface roughness (Sa) on a nanometer level were found among some of the implants investigated and when comparing Sa values, the roughness on the nanometer level did not correspond with the changes of roughness on the micrometer level. Interestingly, some implants show the same mean surface roughness (Sa) on both the micro- and nanometer levels, although SEM revealed specific nanosized structures on one surface, which were not found on the other. These specific nanostructures could not be detected with interferometry, which indicate that the interferometer is not suitable to detect specific nanosized structures, but can be used only to describe the nanometer surface roughness in terms of height deviation. To completely characterize the surface topography of a dental implant on a nanometer level, a complementary examination with SEM is necessary.

In study IV and V the same protocol was used to characterize nanometer roughness as evaluated in study I. There were no statistically significant differences between the surfaces, however the nano-HA surfaces had a slightly lower Sa mean value, maybe indicating that the coating somewhat smoothens the surface on this level of resolution. In study III the interferometry was used to simply verify the absence of microstructures on the cylindrical implants.

methods and results in vitroAdditional surface characterization with AFM should preferably have been performed in study II. The optical trap technique and QCM-D are not standard methods to evaluate cell attachment towards different surfaces, however it was found possible to measure cell attachment using the two techniques. Although, in vitro studies are far from the clinical situation and this should be kept in mind when interpreting results. Further studies are necessary to evaluate

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if the techniques may be used to increase our understanding of the initial interaction between cells and an implant surface and to further evaluate if the techniques may offer predictive tests when evaluating novel implant surfaces.

Optical trapNot many studies have previously been conducted with the aim to compare initial cell adhesion to different surfaces using an optical trap. To attach, the cells needed to be in direct contact with the test surface and in case of attachment, it was not possible to remove the cell from the surface using the tweezers, indicating that the adhering force was stronger than the highest force (40-50pN, depending on cell size) exerted by the trap. The same result was previously reported by Andersson et al.130. Thermal effect from high photon intensities on the cell can be seen in a direct temperature rise. The temperature increase varies between 1-2°C when using laser tweezers with a wavelength of 1064 nm (as the one used in study II) which may affect the cell135. Another issue is the small amount of cell suspension (less than one ml) that is put between the surface samples, which cool down quite quickly and the environment for the cell will therefore not be optimal and may affect the cell behaviour and subsequently the test result. These potential errors should be considered when interpreting the results. QCM-DWhen interpreting data from QCM-D measurements, an increase in frequency is normally related to a decrease in mass. However, the initial increase in frequency during cell adhesion observed in study II was accompanied by a large increase in dissipation. Additionally, cell attachment to the crystal was verified with light microscopy. Therefore, the increase in frequency could not be due to loss of mass. In a study performed by Marxer et al.136 cell spreading was investigated using QCM and positive frequency shifts were found during cell adhesion onto gold coated crystals. Furthermore, Granéli et al.137, found positive frequency shifts with increasing vesicle layers when studying the formation of multiple layers of lipid vesicles on a QCM crystal. The increase in frequency was followed by a large increase in dissipation. The positive frequency shifts observed in the

57

studies may be explained by an acoustic wave. A standing acoustic wave, or film resonance, occurs at certain film thickness and at certain frequency magnitudes, which results in resonance oscillation of the film with the fundamental crystal oscillation. Höök et al.138 suggested that this behaviour can be efficiently used for determining the viscoelastic properties and thickness of polymer coatings. However, it might not be applicable to predicting cell viscoelasticity, due to the more complicated structure of the cell, which makes assumptions regarding density etc. more difficult.

In study II, the increase in frequency was followed by a slow decrease. This is believed to be due to the continuous flow of serum containing cell media over the crystals post cell adhesion, which results in a small adsorption of proteins onto the crystal area not covered by the cells. It should also be noted that the shift in dissipation cannot be explained only by protein adsorption since protein adsorption generally results in dissipation shifts of much smaller magnitudes.

A linear correlation between the magnitude of dissipation increase and the cell surface coverage (CSC) was found for both osteoblasts and fibroblasts. In contrast, the magnitude of frequency did not correlate to the percentage CSC for neither the osteoblasts, nor the fibroblasts (figure 11). Modin et al.139 studied the initial attachment and spreading of pre-osteoblastic cells onto Ta and Cr coated QCM-crystals over a 60 min period. The results demonstrated increase in dissipation but also decrease in frequency with increasing amount of cells injected onto the crystal, which does not correlate with the observations from study II. However, in study II, the measurements continued for at least 16 hours and the large increase in dissipation did not occur until after 60 to 120 min post cell injection, except for the very high CSC where the large increase in dissipation started already after 30 min but did not reach its highest value until after at least 100 min. Furthermore, observations indicate that initial cell adhesion experiments with QCM-D should be monitored for at least 8 hours and that it is the dissipation and not the frequency that reveals the kinetic behaviour of cells adhering and spreading onto a surface. The initial increase in dissipation may be a sign of the cells first adhere and spread on the surface. The following decrease may then be explained by the cells become more rigid as they come in contact with each other and enter another structural stage.

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Figure 12 compare the recorded shifts in dissipation during osteoblast and fibroblast adhesion and spreading onto nano-HA and titanium surfaces. If there was a difference in the initial cell adhesion between the different surfaces investigated, it could not be detected by comparing the dissipation and frequency shifts.

methods and results in vivoAnimals and surgical techniqueThe chosen rabbit model of this thesis has frequently been used and documented in the literature140-142. Rabbit tibia is suggested to mimic the human mandible and rabbit femur the human maxilla with a more heterogeneous bone quality compared to the mandible. However, when interpreting in vivo results, some issues must be taken into consideration; human jawbone is of intramembraneous origin while the bone in rabbit femur and tibia are of endochondral origin. Furthermore, the experimental implants are not dynamically loaded which is the final aim in the clinic. The evaluation times of 2, 4 and 9 weeks chosen in this thesis, represent the early healing from when new bone formation can be expected (new bone may be observed after 2 weeks in rabbit) and the subsequent healing process. However, complete healing in mature bone was not evaluated (at least 12 weeks or more in rabbit)143.

In order to reduce the number of animals, studies IV and V were based on the same 27 rabbits. However, if a complication occurs, it will affect both studies which of course represent a substantial drawback. Five out of the 27 rabbits suffered tibia fracture and had to be sacrificed before the set healing times and these 5 rabbits were not included in any results. An error search was made when the experimental part of the study was completed. Surgery was carried out according to a standard protocol and after well documented procedures with no complications and by an experienced operator. No reason could be found for these fractures.

Removal torque analysisThis method has been described frequently in the literature for biomechanical evaluation of threaded implants. It has been shown that results from removal torque tests correlate with BIC values when evaluating micro surface topography and the test roughly

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reports on the shear strength between the bone and implant. It has been suggested that the removal torque test may be a reliable method for evaluation of bone response towards nanostructured surfaces as well144. In study IV, the results from the removal torque analysis showed a tendency for higher values for the double coated nano-HA after 2 weeks of healing and for both the single and double coated nano-HA surface after 9 weeks of healing but the differences were not significant. No significant differences were found in study V. It may be that the microroughness of the investigated implants are of superior importance to the biological response and the discrete changes in bone response reported in some studies will not be significant in the removal torque test when nanostructures are applied to an already present microroughness.

Histological specimen preparation and evaluationAll histological evaluations were performed in a blinded manner, i.e. the investigator was unaware of which type of surface that was evaluated. The histological samples were prepared according to Donath et al.132 and this is a commonly used method. Bone in contact (BIC) and bone area (BA) measurements are standard procedures when reporting histological results in the literature. Measurements of newly formed bone as used in this thesis, is not standard, although applied in studies IV and V. The information gained from one histological section does not provide the complete picture of the implant integration since it is only two dimensional. In study IV and V, BIC was not evaluated since removal torque tests were performed and thereby the bone to implant contact disrupted. BIC evaluations would have been unreliable.

In study IV the histological evaluations indicated a tendency of more new bone formation to the coated implants compared to the control but the differences did not reach statistical significance, possibly due to the control having nanostructures present, although not in form of HA nanocrystals. In study III, no significant differences in BIC or BA values were found when measured on the lateral walls of the cylindrical implants. However, on the apical part, a significant increase in BIC values were seen for the nano-titania surface, this may be explained by the almost doubled Sdr values for the nano-titania compared to the nano-HA implant.

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According to results from this present thesis; a nano-HA coating demonstrated similar results in vivo as a nano-titania coating and uncoated controls. As previously mentioned, several in vivo studies have been performed on nanostructured surfaces, some of those confirming their importance for osseointegration whereas others like the present thesis have failed in doing so.

Lee et al.145 concluded that an additional coat of HA nanocrystals on screw-shaped ceramic implants do not enhance the early bone response. Furthermore, in an in vivo study in goats, screw-shaped grit-blasted, acid etched (GAE) and electro-sprayed CaP nanoparticle-coated implants gave similar bone responses and torque values as found to GAE alone146. Schliephake et al.147 compared dual acid-etched (DAE) screw-shaped implants with and without a HA coating (using electrochemical deposition) in a dog model. No significant differences in bone contact, or in bone volume density was found. Coelho et al.148 found that a 20-50 nm thick CaP based coating on a blasted and etched cylindrical implants did not improve the biomechanical fixation or BIC after 2 and 4 weeks of healing in dog tibia. As mentioned above, other authors have found positive effects of nanostructures on implant surfaces in vivo. Bjursten et al. 89 compared TiO2 gritblasted surface and TiO2 nanotube discs in the rabbit tibia. The results showed significantly improved bone-bonding strength, increased bone-implant contact and new bone formation to TiO2 nanotubes compared to the TiO2 gritblasted surface. Mendes et al. 82 found higher disruption forces to a dual acid-etched titanium surface (the implants were rectangular plates) with discrete crystalline deposition (DCD) of calcium phosphate (CAP) compared to non-DCD samples when placed in rat, again confirming the importance of nanostructures.

Implant design will probably affect the results; many in vivo studies are conducted on smooth implants with a macro design that may not present with initial implant stability. When nanostructures are deposited on screw-shaped implants already having a defined microroughness, potential positive biological responses to the nanostructures may be masked by the microstructures, assuming the latter representing the dominant surface features. The study design and the use of different species of animals may further affect

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the results, making it difficult to compare one study with another. Another reason for lack of positive results when investigating nanostructures may be that our traditional evaluation techniques are too coarse to detect biological effects on the nanometer level.

The fact that there are different techniques of manufacturing nanostructures, resulting in different size, shape and distribution of these structures may further result in different biological response as reported by Yang et al.74 who compared biomimetically deposited CaP (BDCAP) and electrochemically deposited nano-hydroxyapatite (EDHA) with evidence of EDHA showing significantly improved bone response after 6 and 12 weeks of implantation.

Study IV was performed in cortical bone, which gives the implant good primary stability and perhaps therefore the nanostructures did not give a detectable additional effect in bone response. One hypothesis was that the situation perhaps may be different in trabecular bone, which usually gives less primary stability and is commonly associated with varying bone quality as mentioned above. The positive response reported in the previously mentioned human studies were all conducted in the posterior maxilla which indeed has greater variations of bone quality e.g. density and thickness of the cortical layer. The implants used in those studies were all DAE implants with a DCD of CaP. The nano-HA coated implants in study V performed similarly as the un-coated controls when placed in the rabbit femur that is suggested to mimic human maxilla, unable to prove the importance of nano-HA in early bone healing. It should be considered that micro- and nanotopography as well as chemistry was different, since DAE implants were used in the mentioned human studies and sandblasted/acid etched implants were used in this thesis, further, the DAE were CaP coated while the latter surfaces were coated with nano-HA.

summary and topics for future researchThe overall aim of this thesis was to characterize dental

implants on the nanometer level of resolution; to evaluate whether nanostructures applied on implants without micrometer topography may be important in the initial cell response and early bone healing.

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Another aim was to evaluate whether additional nanostructures on micrometer rough implant may improve the biological response in bone of different qualities.

In vitro results from the literature evaluating nanostructures clearly indicate that different types of nanostructures applied on different surfaces do have an impact on the cell response. However, results regarding the importance of nanostructures in early bone healing and osseointegration are diverse and one cannot with certainty state any conclusions. Some in vivo studies report on significantly improved bone response to nanostructured surfaces while others, in consistency with results from the present thesis, find similar bone response when compared to unmodified controls. Further studies are needed to fully evaluate to what extent nanostructures influence the early bone healing process and osseointegration.

There are different ways of manufacturing nanosized structures, thereby creating different surface topography and chemistry. Studies in vitro and in vivo have shown that altered nanoroughness and different types of nanostructures alter the biological response differently indicating that the importance of different chemistry, sizes, shapes and distribution of nanostructures should be evaluated further.

However, possibly even more important, efforts toward finding a standardized method for characterization of these nanostructures should be developed and related to the biological response and thus be useful as a predictor of the clinical outcome.

One may consider it difficult to further improve the clinical success rates of today’s commercially available implants when placed in healthy bone of good quality and quantity. However, implant surface improvements may be of great benefit to patients with compromised bone, therefore further research should aim to investigate whether nanostructures may influence bone healing in conditions with poor bone quality; whether nanostructures may be of significance in the clinical treatment of patients with focus on their short term as well as long term performance. Furthermore, studies have indicated that nanostructures may have an influence in soft tissue response and bacteria adhesion, issues that may be of clinical importance and therefore should be topics for future studies.

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cOnclusiOns

Surface characterization;• An optical interferometer may be used for detection of

nanoscale roughness in vertical dimension; however, limita-tions exist in the horizontal plane due to the wavelength of the light. To detect specific nanostructures, the inter- ferometer analysis is insufficient and SEM proved to be a good compliment.

The nanometer roughness was not necessarily corre-lated to the micrometer roughness. Different structures in the nanometer scale was found on some of the implants, indicating that to fully understand the relationship between the properties of an implant surface and its osseointegration behaviour, a characterization at the nanometer scale may be relevant.

Real-time in vitro;• It was demonstrated possible to measure cell attachment

using optical trapping and QCM-D, however, no obvious differences in initial osteoblast or fibroblast response to different surfaces could be detected.

In vivo bone response;• Smooth cylindrical titanium implants coated with nano-HA

or nano-titania showed similar early bone responses when evaluated in cortical bone.

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• Nanometer thick coatings of HA nanocrystals did not enhance early bone healing when applied on blasted and etched screw-shaped implants compared to non-coated ones and placed in cortical bone.

• A coating of hydroxyapatite nanocrystals deposited onto blasted and acid etched screw shaped titanium implants, did not enhance bone healing compared to non-coated implants when evaluated in trabecular bone.

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pOpulärvetenskaplig sammanfattning

Dentala implantat används dagligen runt om i världen. Att ersätta tänder med hjälp av titanimplantat är en mycket pålitlig behandling, där lyckandefrekvensen oftast är över 90 % enligt vetenskapliga rapporter. Dagens pågående implantatforskning syftar till att ytterligare kunna förbättra implantatens egenskaper för att förbättra inläkningen hos patienter med sämre ben kvalitet och/eller kvantitet där lyckandefrekvensen är lägre jämfört med patienter där käkbenets status är bra. Vidare syftar forskningen till att hitta en optimal yta för att eventuellt kunna påskynda läkningsprocessen och kunna belasta implantaten tidigare, vilket skulle resultera i kortare behandlingstider. Forskning pågår även för att ytterligare öka förståelsen för de molekylära och cellulära mekanismerna bakom inläkningen av implantat i ben och mjukvävnad. De senaste åren har forskningen riktats mot nanoteknologi och om huruvida nanostrukturer skulle kunna påverka den tidiga implantatinläkningen.

Implantatens design och ytkaraktär har i tidigare forskning visat sig kunna påverka inläkningen i benvävnad. Man kan dela in ytråhet i millimeter, mikrometer och nanometernivå. Hittills finns kunskap om att millimeter- och mikroråhet påverkar det biologiska svaret, men förståelsen om i vilken omfattning nanostrukturer påverkar är begränsad. Man kan på flera sätt modifiera ytan på implantat t.ex. med blästring, syraetsning eller olika ytbeläggningar, vilket resulterar i olika ytråhet men kan dessutom förändra ytkemin. Det har i tidigare studier visats att en medelrå yta, vid utvärdering på mikronivå, inducerar bättre cell och benrespons jämfört med slätare

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och råare ytor. Ytbehandlingar som syftar till att förändra en yta på mikronivå borde även förändra ytkaraktären på nanonivå, vilket kan påverka implantatinläkningen eftersom det är på denna nivå som de första proteinerna binder in till ytan efter implantatinstallation och därefter kommer cellsvaret. Flera cellstudier har visat att olika typer av nanostrukturer påverkar benceller på ett positivt sätt. Även djurstudier har visat att nanostrukturer har en positiv inverkan på benläkningen med ökad benkontakt till implantatytan samt ökade urvridningsvärden vid biomekanisk utvärdering. I vilken utsträckning nanometerstrukturer påverkar inläkningen i ben och mjukvävnad samt huruvida de påverkar den kliniska behandlingen av patienter är i dagsläget inte känt.

Den här avhandlingen har syftat till att undersöka yttopgrafin på implantat med inriktning på nanometertopografi och dess betydelse för inläkningen av titanimplantat i benvävnad.

Ett av syftena med denna avhandling var att hitta en metod för att karaktärisera kommersiella dentala implantatytor på nanometernivå. Resultaten från denna studie visade att kommersiella implantat har nanoråhet av varierande grad, men också att en del av implantaten hade specifika nanostrukturer. Vidare visade resultaten att nanoråheten inte hade någon direkt korrelation till mikroråheten, ett implantat som på mikronivå klassas som slätt var inte nödvändigtvis det på nanonivå. Dessa observationer indikerar att det kan vara relevant att även undersöka implantatytors nanotopografi när man i studier undersöker den biologiska effekten av en implantatyta i cell och djurstudier.

Med hjälp av två tidigare inte så använda tekniker, en optisk pincett samt QCM-D, undersöktes den första interaktionen mellan cell och implantatyta samt om det fanns någon skillnad i cellsvar till olika typer av ytmodifieringar. Resultaten visade att det var möjligt att med hjälp av teknikerna undersöka cell-adhesion men ingen självklar skillnad kunde påvisas mellan de olika ytorna.

Släta och medelråa implantat med olika design och belagda med nanopartiklar av hydroxylapatit (nano-HA) utvärderades vid tre olika inläkningstider för att undersöka om nanostrukturerna kunde

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ha någon positiv effekt på inläkningsförloppet. Vidare utvärderades implantaten i ben av varierande kvalitet. Efter biomekanisk urvridning och analys av histologiska snitt visade resultaten att nano-HA implantaten gav liknande biologisk respons som icke belagda kontrollimplantat. Tidigare djurstudier har visat positiv effekt av nanostrukturer på den tidiga inläkningen i ben. Flertalet av dessa studier är gjorda på icke skruvformade implantat vilket kan ha haft en inverkan på resultatet eftersom designen på implantatet tidigare visat sig vara av signifikans. Det finns även tidigare studier som i likhet med resultaten från denna avhandling, inte kunnat påvisa någon ytterligare positiv biologisk effekt av nanostrukturer på implantat med skruvdesign och en redan befintlig mikroråhet. Man kan med flera metoder skapa nanostrukturerade ytor med olika topografi och kemi. Olika typer och storlekar av nanostrukturer testas och utvärderas på olika sätt i studier vilket kan resultera i varierande biologiskt svar. Detta gör det svårt att jämföra resultaten från studierna samt gör det svårt att dra adekvata slutsatser från forskningen.

Sammanfattningsvis har resultaten i denna avhandling visat att kommersiella dentala implantat har nanoråhet samt specifika nanostrukturer av varierande grad. En standardiserad metod borde utvecklas för att beskriva implantaten på nanonivå för att lättare kunna jämföra studier med varandra och öka kunskapen om nanostrukturers inverkan på implantatinläkningen. Det gick inte fastställa någon skillnad i initialt cellsvar mellan olika typer av ytmodifieringar när det undersöktes men med en optisk pincett eller QCM-D. En ytterligare beläggning av nano-HA på släta cylindriska implantat gav liknande beninläkning som en beläggning av nano-titan på samma typ av implantat. Implantat skruvar med en medelrå mikroyta och en ytterligare beläggning av nano-HA gav liknande beninläkning som icke belagda kontrollimplantat när de testades i ben av olika kvalitet. Ytterligare studier behövs för att klargöra i vilken utsträckning nanostrukturer kan påverka den tidiga inläkningen av benförankrade implantat samt om nanostrukturer kan vara av signifikans i den långsiktiga behandlingen av patienter.

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acknOWleDgements

My sincere gratitude and deepest thanks to all that have contributed to the work of this thesis. I would especially like to thank the following;

Professor Ann Wennerberg, my brilliant supervisor. For introducing me to the world of science and for sharing your expertise. For always being supportive and caring in every matter. I am more than glad and grateful to have been your student. From the bottom of my heart, thank you!

Martin Andersson, my co-supervisor, for support and for patiently guiding me through this work and sharing your knowledge in science. My greatest thanks and appreciation for yours and Maria’s hospitality in Florida as well as in Gothenburg.

Professor Tomas Albrektsson, for always kindly being helpful in any matter. And for giving me the opportunity to begin my PhD studies at the department of Biomaterials in Gothenburg. I gratefully appreciate your support.

My greatest thanks to all my co-authors for sharing your knowledge and for your valuable contributions to this work; Maria Hoffman, Emma Westas, Luis Meirelles, Viktoria Franke-Stenport, Kamal Mustafa, Fredrik Currie, Per Kjellin, Ling Wang, Randy Duran, Timo Peltola and Ikka Kangasniemmi.

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Thanks to everyone at the department of Prosthodontics in Malmö for creating a good work environment. Ulf, for always being helpful in administrative matters.

Thanks to the involved persons at the department of Biomaterials in Gothenburg, in particular for Petra and Ann for all help with preparing histological samples. Maria H. and Anna W. for always being helpful, inspiring and encouraging.

Thanks to Lennart Carlsson for kindly preparing surfaces.

Birgitta Gunnarsson, my first boss and mentor at the Selma clinic in Gothenburg. Thank you for from the start of my work as a dentist believing in me, for never failing support and friendship. You are an inspiration and a role model and you have influenced my way of thinking.

Friends and colleagues at the Colosseum Clinic in Halmstad, thank you all for making every day at work joyful. In particular, Kicki, my right hand, for always encouraging talks and your great sense of humour. I am so glad to have you as my friend.

To all in my dear family and my friends for always believing in me even when I do not and for making my life joyful. In particular;

Pelle, for being there with support and believing in me, for such a long time. For patiently helping me back after my accident. Without you I would not be where I am today.

Valle and Marie, for computer support, for many good and crazy memories and for reminding me of the joyful things in life. I really treasure our friendship.

Simon, you truly are very good friend. For your never failing support and encouragement, in good times and in bad. I am grateful to have you in my life.

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My lovely mother Nina, for tremendous support and always being helpful. I appreciate everything you do. From the bottom of my heart dearest mom, thank you.

My father Allan, thank you for always believing in me and for your constant support in everything I do. My dear grandmother Millan and grandfather Olle, I admire your strength and I have the deepest respect for you both. Thank you for always believing in me and at the same time always giving the feeling that you love me for who I am.

At last, but for sure not least, my son Sammy, the sunshine in my life, my everything!

This work was supported by grants, here gratefully acknowledged from; Wilhelm and Martina Lundgren Science Foundation, Hjalmar Svensson Foundation, the Sylvan Research foundation, Sigge Perssons & Alice Nybergs stiftelse (Gothenburg Dental Society), the Swedish Dental Society, the Swedish Research Council and the Swedish Knowledge Foundation.

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