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O n t i s s u e r e ac t i O n s tO a n d r e s O r p t i O n O f b O n e s u b s t i t u t e s

Malmö University Faculty of Odontology Doctoral Dissertations 2013

© Arne Mordenfeld 2013

Photographers: Arne Mordenfeld, Eva Larsson, Karin Edkvist Jonsson

ISBN 978-91-7104-393-1

Service Point Holmbergs, 2013

arne MOrdenfeld On tissue reactiOns tO and resOrptiOn Of bOne substitutes

Department of Materials Science and Technology, Malmö University

Department of Oral & Maxillofacial Surgery, Gävle county hospital

Department of Biomaterials, The Sahlgrenska Academy, University of Gothenburg, Sweden, 2013

This publication is also available at:www.mah.se/muep

To my mother and father – you would have been proud

“Somewhere, something incredible is waiting to be known”

Prof. Carl Sagan

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This thesis represents number 44 in a series of investigations on implants, hard tissue and the locomotor apparatus originating from the Department of Biomaterials, University of Gothenburg and the department of Prosthodontics/Material sciences, 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. Fredric 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 Res Tech, 1991. On Tissue Reaction 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. Davies.

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.

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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 RCSm 1997. On the Clinical Measurement of Implant Stability 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 Patellfemoral 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 Impant 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 Johansson MD, 1999. On Implant Integration in Irradiated Bone. An Experimental Study of the Effects of Hyperbaric Oxygeneration and Delayed Implant Placement. Thesis defended 8.12.1999. External examiner: Docent K. Arvidsson-Fyrberg.

22. Börje Svensson FFS, 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 on Total Hip Arthroplasties: Pre-Clinical Evaluations. Thesis defended 1.9.2000. External examiner: Dr A.J.C. Lee.

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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 experi-mental implants. Thesis defended 19.11.2001. 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 CCS, 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 Chemomechanival 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: Professor B. Melsen.

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35. Andreas Thor DDS, 2006. On platelet-rich plasma in reconstructive dental implant surgery. Thesis defended 8.12.2006. External examiner: Professor 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: Professor 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 K. Arvidsson 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: Professor K. Arvidsson 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 defended 01.06.2011. External examiner: Associate professor C. Dahlin.

42. Byung-Soo Kang Msc, 2011. On the bone tissue response to surface chemistry modifications of titanium implants. Thesis defended 30.09.2011. External examiner: Professor J. Pan.

43. Kostas Bougas DDS, 2012. On the influence of biochemical coating on implant bone incorporation. Thesis defended 12.12.2012. External examiner: Professor T. Berglundh.

44. Arne Mordenfeld DDS, 2013. On tissue reactions to and resorption of bone substitutes. Thesis to be defended 29.5.2013. External examiner: Professor C. Dahlin.

table Of cOntents

LIST OF PAPERS ........................................................... 13

ABSTRACT .................................................................. 15

POPULÄRVETENSKAPLIG SAMMANFATTNING ..................................................... 19

ABBREVIATIONS .......................................................... 21

INTRODUCTION .......................................................... 23Bone biology .......................................................................24Bone cells and their functions .................................................25Bone formation and remodelling ...........................................27Different types of graft materials .............................................29Graft healing .......................................................................34Surgical techniques ...............................................................39

AIMS ......................................................................... 43General aim ........................................................................43Specific aims .......................................................................43

HYPOTHESES .............................................................. 45

MATERIALS AND METHODS ........................................... 47Subjects and experimental outlines .........................................47Ethical considerations ...........................................................49Preoperative examinations, inclusion and exclusion criteria .......50Grafting materials.................................................................50Dental implants ....................................................................52Drop-outs .............................................................................52Surgical procedures ..............................................................53

Clinical follow up .................................................................58Histology and histomorphometry ............................................61Statistics ..............................................................................63

RESULTS ..................................................................... 67Clinical results ......................................................................67Radiographic results .............................................................70Histology and histomorphometric results ..................................73

DISCUSSION ............................................................... 81Paper I ................................................................................81Paper II ...............................................................................84Papers III and IV ...................................................................86Paper V ...............................................................................91Clinical applications and future perspectives ............................94

CONCLUSIONS ........................................................... 97

ACKNOWLEDGEMENTS ............................................... 99

REFERENCES ..............................................................103

PAPERS I – IV ..............................................................123

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list Of papers

This dissertation is based on the following papers, which will be referred to in the text by their Roman numerals (papers reprinted by kind permission of journal editors):

I. Mordenfeld A, Hallman M, Lindskog S. Tissue reactions to subperiosteal onlays of demineralized xenogenous dentin blocks in rats. Dent Traumatol. 2011 Dec;27(6):446-51.

II. Lindgren C, Mordenfeld A, Johansson C.B, Hallman M. A 3-year clinical follow-up of implants placed in two different biomaterials used for sinus augmentation. Int J Oral Maxillofac Implants. 2012 Sep;27(5):1151-62.*

III. Mordenfeld A, Albrektsson T, Hallman M. A 10-year clinical and radiographic study of implants placed after maxillary sinus floor augmentation with an 80:20 mixture of deproteinized bovine bone and autogenous bone. Clin Implant Dent Relat Res. 2012. Epub ahead of print. DOI: 10.1111/cid.12008

IV. Mordenfeld A, Hallman M, Johansson C.B, Albrektsson T. Histological and histomorphometrical analyses of biopsies harvested 11 years after maxillary sinus floor augmentation with deproteinized bovine and autogenous bone. Clin Oral Implants Res. 2010 Sep;21(9):961-70.

V. Mordenfeld A, Johansson C.B, Albrektsson T, Hallman M. A randomized and controlled clinical trial of two different compositions of deproteinized bovine bone and autogenous bone used for lateral ridge augmentation. Clin Oral Implants Res. 2013. Epub ahead of print. DOI: 10.1111/clr.12143

*This publication has previously been included in a thesis entitled “On healing of titanium implants in biphasic calcium phosphate”, presented for Doctor of medicine degree, defended by Christer Lindgren at Linköping University 2012.

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abstract

Background: The increasing need for bone grafting procedures in implant dentistry and the introduction of a variety of bone substitutes require a deeper understanding of the biological response and short- and long-term behaviour of these materials to choose the adequate graft and surgical procedure for the intended clinical application.

Aims: The overall aim was to clinically and histologically study the short- and long-term tissue reactions to and resorption of bone substitutes after bone augmentation.

Material and methods: In paper I, dentin blocks with different demi-neralization times were placed subperiostally in 40 rat skulls. After a healing period of 4 weeks the rats were sacrificed and the healing of the dentin blocks were evaluated. In paper II, eleven patients were treated with bilateral sinus floor augmentation using biphasic calcium phosphate (BCP) on one side and deproteinized bovine bone (DPBB) on the contralateral side, acting as control. After 3 years, biopsies were retrieved from the grafted area for histological evaluation and histomorphometry and 62 dental implants, placed 8 months after graft healing, were clinically evaluated. In paper III and IV, fourteen (22 sinuses) of the included 20 patients (30 sinuses) treated with sinus floor augmentation with a mixture of 80% DPBB and 20% autogenous bone (AB) from the chin were followed throughout the 10 years study period. These patients had 53 implants placed in grafted sites and 15 implants placed in non-grafted bone. Clinical and radiographic examinations were performed. Biopsies were retrieved from the grafted sinuses after 11 years of graft healing for

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histological evaluation and histomorphometry. The particle sizes were compared with samples retrieved after 6 months from the same patients and pristine particles from the manufacturer. In paper V, 13 patients (14 jaws) were treated with lateral ridge augmentation using 2 different mixtures of DPBB:AB (90:10 and 60:40) in a randomized and controlled trial, designed as a split mouth study. The width and volume changes were evaluated after 7.5 months by means of cone beam computed tomography. After 8 months of graft healing, at the time of implant placement, biopsies were retrieved for histological evaluation and histomorphometry.

Results: Resorption increased with increasing degree of demineraliza-tion of dentin blocks while bone formation increased with increasing degree of demineralization, in the latter case provided inflammation was compensated for (paper I). After 3 years of healing the BCP particles showed different levels of dissolution, in contrast to DPBB particles that showed no signs of resorption. The overall implant survival rate was 96.8% and the success rate for implants placed in BCP and DPBB was 91.7% and 95.7% respectively (paper II). The cumulative survival rate of the implants after 10 years was 86% and the marginal bone loss was 1.6 mm. There was only a reduction in graft height between 3 months and 2 years but no further reduction up to 10 years (paper III). There was no difference between the size of DPBB particles after 11 years compared to those measured after 6 months or to particles from the manufacturer (paper IV). The gain in width of the alveolar crest was 3.5 mm and 2.9 mm and the reduction of the grafts were 37% and 47% for the 60:40 mixture and 90:10 mixture respectively (significant differences). There were no histomorphometrical differences between the groups (paper V).

Conclusions: Partial demineralization may provide a method for optimizing the integration of dentin onlays. A similar degree of bone formation and bone-to-graft contact for BCP and DBB was found 3 years after maxillary sinus augmentation with similar success rates for implants placed in both grafting materials. At 10 years follow-up after sinus floor augmentation with 80:20 (DPBB:AB) graft, the remaining implants presented good clinical and radiological results and there seems to be no further graft resorption after 2 years of

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graft healing. DPBB particles were found to be well integrated in lamellar bone, showing no apparent signs of resorption after 11 years in humans. Despite a small difference in width changes after lateral ridge augmentation, the amount of AB added to DPBB did not seem to have a major impact on the graft healing and graft reduction, thus making it possible to install implants in all grafted sites.

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pOpulÄrVetensKapliG saMManfattninG

Tandlöshet och tandprotesbärande innebär ofta ett stort socialt handikapp. Idag är tandimplantat en väl etablerad metod för att ersätta tandförluster. I vissa fall saknas tillräcklig benmängd för att kunna installera implantat på ett adekvat sätt eller på rätt position för att uppnå ett fullgott estetiskt resultat. Transplantat med ben från annan del av patientens käkar eller från höften har varit den mest accepterade metoden för att bygga upp det förlorade benet efter tandförluster. Detta ingrepp innebär ytterligare kirurgi, som inte sällan måste utföras i narkos, med ökade besvär för patienten. För att minska patientens besvär och sjukvårdens kostnader har benersättningsmaterial blivit allt vanligare både inom käkkirurgin och i den ortopediska verksamheten. Det ökade behovet av bentransplantationer på senare år och introduktionen av nya material på marknaden ökar vikten av kunskap om hur materialen fungerar kortsiktigt och långsiktigt. Det är angeläget att minimera ogynnsamma vävnadsreaktioner och att välja rätt material i det individuella fallet för ett optimalt slutresultat.

Denna avhandlings övergripande syfte var att kliniskt och histo-logiskt utvärdera vävnadsreaktioner efter transplantation med dentin (tandben), ett syntetiskt benersättningsmaterial (keramiskt material) och speciellt behandlat kalvben, både kortsiktigt och långsiktigt, samt att bedöma materialens eventuella resorption.

I den första studien utvärderades möjligheten att påverka benin-läkningen vid transplantation (till råttor) av dentin med olika urkalkningsgrad.

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I studie II utvärderades vävnadsreaktionerna och materialens resorption 3 år efter transplantation av syntetiskt benersättnings-material och kalvben till bihålorna för att öka benmängden inför behandling med tandimplantat. Dessutom utvärderades tandimplan-tatens 3-års överlevnad och status.

I studie III och IV utvärderades vävnadsreaktionerna och materialets resorption 10-11 år efter transplantation med kalvben till bihålorna och tandimplantatens överlevnad samt status.

I studie V utvärderades betydelsen av olika mängd tillsatt eget ben till kalvben för transplantatets inläkning vid uppbyggnad av extremt tunna käkben och volymstabilitet inför installation av tandimplantat. Studierna visar att:

• Partiell urkalkning kan vara en fungerande metod för att optimera beninläkningen av dentin.

• Det var ingen skillnad i beninläkning efter 3 år i bihålan mellan kalvben och syntetiskt benersättningsmaterial. Det senare uppvisade dock en högre grad av inflammation. Lyckande- graden för tandimplantat var lika hög i båda transplantaten (96%).

• Det tycks inte föreligga några volymsförändringar av kalvben mellan 2 och 10 år efter transplantation till bihålan. Tandimplantaten uppvisade goda kliniska och radiologiska 10-års resultat oavsett om de var placerade i transplanterat eller eget ben.

• Kalvbenet var mycket väl inläkt 11 år efter transplantation till bihålan och kalvbenspartiklarna uppvisade inga uppenbara tecken på resorption.

• Det var lika god inläkning av transplantaten om det hade tillsatts 40% eller 10% eget ben till kalvbenet vid uppbyggnad av tunna käkar. Det var något mindre breddvinst med 10% eget ben, men i samtliga 14 fall lyckades man installera tandimplantat 8 månader efter benuppbyggnaden oavsett vilken transplantatblandning som använts.

• Det finns idag benersättningsmaterial på marknaden som fungerar väl vid uppbyggnad av käkben inför implantatbehandling och sålunda minskar både patientens lidande och kostnaderna för sjukvården.

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abbreViatiOns

AB Autogenous boneBCP Biphasic calcium phosphateBMP Bone morphogenetic proteinBMU Basic multicellular unitBoP Bleeding on probingCaP Calcium phosphateCB/CT Cone beam computed tomographyCSR Cumulative survival rateDPBB Deproteinized bovine boneEDTA Ethylene-diamine-tetra-acetic acidGBR Guided bone regenerationHA HydroxyapatiteIGF Insulin-like growth factorIL InterleukinISQ Implant stability quotientM-CSF Macrophage colony-stimulating factorMGC Multinucleated giant cellsMMP Matrix metalloproteinaseOB OsteoblastOC OsteocyteOPG OsteoprotegerinOPN OsteopontinPD Pocket depthPI Plaque indexPMN Polymorphonuclear neutrophilPTH Parathyroid hormoneRANK Receptor activator of NF-κβ

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RANKL Receptor activator of NF-κβ ligandRFA Resonance frequency analysisRGD Arginine-glycine-aspartic acidSBI Sulcus bleeding indexTBV Total bone volumeTCP Tricalcium phosphateTGF Transforming growth factorTRAP Tartrate resistant acid phosphatase

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intrOductiOn

The loss of teeth is commonly caused by caries, periodontal disease, trauma and infection. Edentulous or partially edentulous people may have aesthetical, social and functional problems. Implant supported prosthesis is a procedure that successfully has been used as an alternative treatment to fixed dental supported bridges and removable dentures in the past 30 years with good results.1 As a consequence of tooth loss the alveolar bone is resorbed in various degrees,2-4 often resulting in inadequate bone volume to allow dental implant placement or restricting ideal positioning of the dental implants. In addition to resorption of the alveolar crest, inadequate bone volume is also caused by pneumatised sinuses of the posterior maxilla.5 When there is a lack of bone height or bone width, bone grafts may be harvested from the jaws or the iliac crest and grafted to the area in need of bone reconstruction. After a period of graft healing, implants can be successfully installed in the reconstructed area. Autogenous (from the same person) bone (AB) has been considered the “gold standard” in bone reconstruction. However, this procedure is associated with morbidity at the donor site,6 prolonged operating time, higher costs, sometimes a need of general anaesthesia and the subsequent degree of graft resorption may be difficult to predict.7-9

In order to reduce morbidity and to simplify the procedure bone substitutes have been used instead of or in combination with AB. Due to higher aesthetical demands combined with an increase of dental implant treatment, the need for bone grafts and bone substitutes has dramatically increased in recent years.

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Some authors suggest that the ideal bone substitute should be biocom-patible, resorbed and replaced by new bone,10 and should possess properties that may enhance bone graft incorporation (i.e. osteoinduc-tive and osteoconductive characteristics)11 without inducing unwanted side effects such as excess inflammation-induced resorption.

As a result of tooth avulsion and injury to the periodontal membrane the root may be replaced by bone (ankylosis) after replantation.12 Furthermore, it has been suggested that dentin possesses both osteoinductive (contains BMP) and osteoconductive properties. These facts may indicate that dentin might function as a bone substitute even if some results from animal studies are cont-radictory.13-15 Further development of the pre-operative preparation of the dentin material and optimizing suitable osteoconductive environment may enhance graft integration.

One of the most documented bone substitutes for intra oral augmentation is Bio-Oss® (Geistlich, Wolhusen, Switzerland), a deproteinized and sterilized bovine bone.16-18 Since all proteins are removed from the material, AB is often combined with BioOss® when grafted to the jaws to add osteoinductive properties. Further clinical and histological studies are necessary to investigate the fate of Bio-Oss® grafts in the long-term. In addition, it is not known if the combination of Bio-Oss® and AB contributes to a better healing and less resorption in lateral ridge augmentation.19

Since biological bone substitutes have a potential risk of disease transmission.20, 21 there has been a growing market for synthetic bone substitutes. Several synthetic bone substitutes have been used for sinus floor augmentation.22, 23 Recently a novel biphasic calcium phosphate (BCP), (BoneCeramic®, Straumann, Basel, Switzerland), was introduced to the market, however long term clinical and histo-logical studies of this graft material are missing.18

The increasing need for bone grafting procedures in the recent years and the introduction of a variety of bone substitutes requires a greater understanding of the biology of bone and bone grafts to choose the adequate graft and surgical procedure for the intended clinical application.

bOne biOlOGyBone biology developments in recent years have nicely been presented in a review by Mackiewicz et al.24 The skeleton of the

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human body is composed of 213 bones and make up about 20% of the body mass.24 The bones can be categorized as long bones (i.e. humerus, femur and tibia), short bones (i.e. carpals and tarsals), irregular bones (i.e. mandible, vertebrae and sacrum) and flat bones (i.e. scapula, sternum and the scull). Bones have several functions including support and protection of vital organs; attachments for muscles, ligaments and tendons; locomotion; support of haemato-poiesis in bone marrow; reservoir for and regulation of minerals (in particular calcium); and repair of bony defects and fractures.25,

26 The bones are composed of cortical or compact bone (80%) and trabecular bone (20%). Cortical bone is mainly found in the surface of flat bones and in the shaft of long bones. The cortical bone has an outer layer called periosteum, consisting of an outer and inner fibrous layer, whose osteogenic potential is important for apposi-tional growth and fracture healing. The inner surface of the cortical bone and the surfaces of the trabecular bone are covered by the endosteum. The trabecular bone accounts for about 70% of the bone metabolism and ensures the stability and elasticity of the skeleton.24,

27 Fifty to 70% of the bone is composed of mineral, mainly hydroxy-apatite, Ca10(PO4)6(OH)2), and small parts of magnesium, carbonate and acid phosphate. The bone matrix (20% to 40% of the bone) consists of type I collagen (90%) and non-structural fibres like osteocalcin, bone sialoprotein, osteopontin (OPN), osteonektin and a great number of growth factors e.g. bone morphogenetic proteins (BMPs), insulin-like growth factors (IGFs) and transforming growth factor-β (TGF-β). The remaining components consist of 5% to 10% water and < 3% of lipids.24

bOne cells and their functiOnsThere are three types of bone cells defined which are responsible for the bone turnover and healing of fractures: osteoblasts, osteocytes and osteoclasts.

Osteoblasts (OB)The primary function of the OBs is bone formation. They are derived from the multipotential mesenchymal stem cells alternatively capable of differentiating into fibroblasts, chondrocytes, myoblasts and adipocytes. In osteoblast differentiation, 4 stages of maturation

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have been identified according to Kular and co-workers28; preosteob-last, osteoblast, osteocyte and bone-lining cells (non-active flattened osteoblasts). OBs secrete type I collagen and produce non-collage-nous proteins including osteocalcin and alkaline phosphatase, which is responsible for mineral deposition. Approximately 50% to 70% of OBs undergo apoptosis and the rest become bone-lining cells or osteocytes. Macrophage colony-stimulating factor (M-CSF), osteo-protegerin (OPG) and cytokine receptor activator of NFκβ ligand (RANKL), factors essential for osteoclast formation and function, are produced by OBs.28

OsteocytesAn osteocyte is a cell located in the bone matrix. The osteocytes represent 95% of all bone cells. They are often regarded as terminally differentiated osteoblasts entrapped and incorporated initially in osteoid (the non-mineralized protein layer between the osteoblasts and the mineralized bone) and then, after mineralization, in more mature, mainly cortical bone. The osteocytes are smaller than osteoblasts and lie in lacunae, spatially separated from each other; however, dendritic processes that extend from their surfaces and traverse the bone within canaliculi connect them with each other as well as bone-lining cells and osteoblasts on the bone surface.28,

29 There seem to be an evidence based fact that osteocytes act as mechano-sensors in bone and control bone formation. In addition, recent research suggest that osteocytes also may play an important role in bone resorption by providing the majority of RANKL that controls osteoclast formation in cancellous bone,30, 31 i.e. osteoblasts/osteocytes share a combined action mechanism with osteoclasts.

Osteoclasts (OC)OC is the only cell type that can resorb bone. They are members of the monocyte macrophage lineage, derived from the hematopoietic stem cells, and are formed via multiple cellular fusions from the mononuclear precursors. A differentiated human osteoclast contains approximately five to eight nuclei. After starting resorption, the osteoclasts have a limited life-span, however the exact mean age is not known, and finally they die via apoptosis.32 Therefore, they must be continuously recruited in the vicinity of the bone resorption

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area. OCs have a unique capacity to dissolve bone mineral and in the end of the resorption process they also perform an enzymatic degradation of the organic bone matrix.32 The osteoclast precursors express receptor activator of NFκβ (RANK). Osteoclast formation, activation and resorption are regulated by the ratio of RANKL (which binds to RANK and activates osteoclastogenesis) to OPG (which inhibits osteoclastogenesis), Interleukin-1 (IL-1) and Interleukin 6 (IL-6), M-CSF, parathyroid hormone, 1,25-dihydroxy-vitamin D (1,25(OH)2-D3), and calcitonin.33, 34 A prerequisite for the OCs to start the resorption process is the attachment to the bone matrix and formation of a sealing zone.35 Osteoblasts secrete matrix metalloproteinases (MMPs) which degrades the osteoid, lining the bone surface, and expose matrix proteins containing arginine-glycine-aspartic acid (RGD) motifs. These adhesion sites are necessary to facilitate osteoclast attachment.36 OCs bind to bone matrix peptides via integrin receptors in the osteoclast membrane.29 The main integrin receptor mediating this osteoclast binding is ανβ3

integrin. The ligands for ανβ3 integrin include osteopontin and bone sialoprotein.24, 36 Once attached to the bone surface, the OC forms a ruffled boarder on the surface of the cytoplasm facing the bone. Secretion of hydrochloride acid starts in the compartment beneath the OCs called resorption lacuna, creating a low pH and subse-quently dissolves the hydroxyapatite. In addition, matrix degrading proteases, mainly cathepsin K, TRAP and MMPs are secreted to digest the bone matrix.32

The induction of bone resorption is mainly regulated by indirect mechanisms. These involve up regulation of the expression of M-CSF and RANKL by osteoblasts, osteocytes and other cells. In addition to the capacity of bone resorption, OCs also regulate osteoblast functions.24

bOne fOrMatiOn and reMOdellinG Two types of bone formation have been described: endochondral ossi-fication (the most common mechanism of primary bone formation) and intramembranous ossification. The endochondral ossification occurs in the long bones, skull base, vertebral column and pelvis. The ossification is preceded by a cartilage template. The cartilage is matured involving hypertrophy of the chondrocytes leading to

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matrix erosion. The remaining cartilage matrix mineralizes, the chondrocytes regress and dies, and the calcified cartilage model is invaded by blood vessels carrying primitive mesenchymal stem cells which may be differentiated into osteoblasts and subsequently start bone formation.37

In intramembranous ossification, bone is synthesized initially without the mediation of a cartilage phase.38 The flat bones of the skull, the clavicle and the mandible are formed through intra-membranous ossification. The primitive mesenchymal cells initially condense into a rich vascularized connective tissue layer. The diffe-rentiation of primitive mesenchymal cells into osteoblasts starts and a trabecular pattern of early bone matrix is produced. Bone matrix is matured through cellular synthesis and secretion of bone matrix components. Thereafter, calcium phosphate in the form of hydroxy-apatite (HA) crystals is deposited at the bone matrix site.37

During adult life old bone, with a high prevalence of micro fractures, is continuously replaced by new bone. Under normal physiological conditions equal amount of bone is formed as the amount of bone resorbed, keeping the total bone mass unchanged. This process is called bone remodelling and is aiming at the maintenance of skeletal mechanical properties and support mineral homeostasis.39 The duration of the resorption process is approxima-tely 3 to 4 weeks and the duration of the following bone formation is approximately 2 to 4 months. Every year approximately 10% of the skeleton (25% of the trabecular and 3% of the cortical bone) is replaced by new bone, thus after 10 years the whole skeleton is renewed.39 Three calcium-regulation hormones control serum calcium: 1,25(OH)2-D3 and; parathyroid hormone (PTH), which stimulates bone resorption, and; an inhibitor of osteoclast resorption, calcitonin.34, 39 Bone remodelling is a complex process starting with the creation of bone resorption cavities by osteoclasts and continues with bone formation by osteoblasts tightly coupled within a basic multicellular unit (BMU). Concentric lamellae of bone in the walls of the cavities are produced by the osteoblasts forming osteons. Four distinct phases can be distinguished in this process: activation, resorption, reversal and formation.27

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different types Of Graft MaterialsThe terminology of different types of bone grafts is based on the genome of the donor and the recipient’s immune-surveillance as described by Urist.40 The term graft applies to viable bone and implant applies to non-viable bone or synthetic bone substitute materials.40

Autogenous bone (AB) graftAB grafts originates from the same individual and is considered “gold standard” in reconstruction of defects in the jaws due to the osteoinductive and osteoconductive properties41 in conjunction with the absent risk of disease transmission and minimal cost. The most common donor sites in pre-implant reconstruction of the alveolar crest and the sinus floor are the mandibular retromolar area, the mandibular ramus, the chin and the iliac crest.16, 42-45 AB has been used extensively for horizontal and vertical augmentation of the alveolar crest as well as for sinus floor augmentation.45-49 AB can be grafted as a bone block or in particulated form using either cortical, cancellous or cortico-cancellous grafts depending on the mechanical stability and volume needed. Particulated bone grafts have mainly been used in small peri-implant defects such as dehiscenses and fene-strations associated with guided bone regeneration,50, 51 but also in larger defects and larger edentulous areas.48, 52 Gordh and Alberius53 reported some basic factors essential to block graft persistence: (i) Embryonic origin, membranous bone seem to be superior over enchondral bone probably due to difference in revascularization and biological difference; (ii) revascularization, incorporation, remodelling and maintenance of the onlay bone graft is related to the rate of revascularization; (iii) graft structure, the cortico-cancellous ratio and the anatomical structure of the cancellous part of the graft seem to influence the rate of revascularization and subsequently determine graft volume persistence; (iv) rigid fixation, is believed to decrease the rate of graft resorption and enhance graft integration.54

Bone substitutesIn this context, bone substitutes are defined as non-autogenous bone grafts including allogeneic grafts/implants, xenogeneic grafts/implants and alloplastic grafts. It has been suggested that the optimal

30

bone substitute should possess osteoinductive, osteoconductive and osteogenetic properties in combination with no risk of transferring infectious diseases. Furthermore, bone substitutes ought to undergo remodelling, be readily available at an acceptable cost, manageable, biocompatible and bioresorbable.55

Allogeneic graft/implantAllogeneic bone originates from another individual of the same species. All of the components in bone, i.e. cells, collagen, ground substances and inorganic minerals, are potentially immunogenic. Bone minerals do not seem immunogenic and collagen is only weakly antigenic. During resorption of the old matrix, allogeneic implants are revascularized by invasion of capillary sprouts from the host bed. Transplantation of allogeneic cortical and cancellous bone leads to an immunologic response, which delays graft incorporation, revascularization, resorption and appositional bone formation.56 In order to sterilize and minimize the immunogenic properties, the graft may be processed by freeze-drying, demineralization, deep freezing (< -70°C), chemo sterilization or radiation. Cortical bone is preferable due to its high content of collagen compared to cancellous bone, resulting in a weaker immunologic reaction. Puros® (MCBA; Zimmer Dental GmbH), a solvent-dehydrated, limited-dose, gamma-irradiated source of allogeneic human bone57 is an example of an allogeneic implant available on the market. Xenogeneic graft/implantXenogeneic bone grafts originate from another species than the host. Furthermore, bone-like minerals derived from corals or algae may be included among the xenogeneic bone substitutes58. The organic portion is removed to obtain safety of disease transmission and to avoid immunologic rejection. Algipore® (Dentsply Friadent, Mannheim, Germany) is a biologic HA derived from calcified maritime algae and the organic components are completely removed by hydrothermal conversion of the calcium carbonate in the presence of ammonium phosphate at about 700°C.59 Algipore® has been used in periodontal treatment60 and for sinus floor augmentation.61

Deproteinized bovine bone (DPBB) is a natural bone mineral, characterized by its high structural and chemical similarity to bone.

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The most documented DPBB used for augmentation in the maxillo-facial region is Bio-Oss® (Geistlich, Wolhusen, Switzerland).

Bio-Oss® has been investigated extensively in both experimental62-65 and clinical studies.18, 22, 66-75 Since all proteins are claimed to have been extracted, Bio-Oss® would not possess any osteoinductive properties and osseous integration of the graft is only achieved by osteoconduction.76 In order to enhance graft integration by osteoin-duction, AB has been added to the graft, in different ratios, in sinus floor augmentation procedures and in alveolar ridge augmentation procedures.71, 77-80 In a recently published systematic review assessing implant treatment outcome when Bio-Oss® alone or Bio-Oss® was mixed with AB used as graft for sinus floor augmentation, Jensen et al71 concluded that the hypothesis of no differences between the two modalities could neither be confirmed nor rejected. The researchers also summarized that addition of AB to Bio-Oss® did not seem to influence the biodegradation of Bio-Oss®, although long-term studies were not available. However, in a recent animal study the same authors concluded that the graft volume was better preserved when DPBB was added to AB grafts and the volumetric reduction of the graft was significantly influenced by the ratio of DPBB and AB after sinus floor augmentation.63 In addition, it appears that the ratio of DPBB and AB may influence the bone remodelling patterns.69 On the other hand, in a randomized controlled trial, it was demonstrated that addition of AB to DPBB in a ratio 1:4 did not lead to enhanced new bone formation in comparison to 100% DPBB, 4 months after sinus floor augmentation.81 Hence, there is a need to further investigate the influence of the AB to DPBB ratio on graft integration and volumetric resistance in clinical, randomized and controlled trials.

There is a controversy in the literature whether DPBB is resorbable or not. Some authors have claimed that DPBB probably is non resorbable,58, 77, 82, 83 however several authors have observed signs of resorption i.e. resorption lacunas and the presence of osteoclasts on the particle surface, or a decrease over time of the fraction of DPBB, in the biopsy, compared to the initial fraction of bone and soft tissues in the graft.17, 65, 84-93 It is of great interest for the clinician to understand the long term outcome of the grafting material, especially when used in aesthetically demanding areas, hence, further investi-gations are necessary to evaluate the long term stability of Bio-Oss®.

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It has been claimed by some researchers that Bio-Oss® after all contains considerable amounts of protein94 and even transforming growth factor β (TGF-β),95 whilst others have questioned these results and found no evidence for the presence of protein in Bio-Oss®96. However, a recent systematic review indicates that bovine derived bone substitutes may carry a risk of prion transmission to patients20 even though no clinical reports of this complication have been published.

Alloplastic graftsAlloplastic bone grafts are chemically produced materials such as calcium phosphate ceramics (CaPs) including HA and tricalcium phosphate (TCP), calcium-sulphate, bioactive glasses and polymers. CaPs are defined as an inorganic phase solid prepared by thermal treatment (sintering) and subsequent cooling.55 The materials can be manufactured in a variety of shapes, forms, structures and chemical compositions. They possess different mechanical and biological properties and the time of degradation may vary. Their similarity in composition to bone mineral, their biodegradability and osteo-conductivity were the rationales for development of CaPs.97 HA is the least soluble of the naturally occurring calcium phosphate salts and resistant to physiologic resorption, whilst TCP has a higher degradation rate. However, the chemical impurity in the contents of the TCP ceramics results in varying degradation times 98 which may lead to unpredictable clinical results. β -TCP has been developed to achieve a more organized crystal structure, still with extensive degradation, and is now frequently used in bone reconstructive surgery. The dissolution process results in high extracellular concen-trations of calcium and phosphate, resulting in precipitation of apatites on a substrate ceramic, forming a carbonate-apatite-crystal layer. This layer is believed to influence the very strong interface between the material and bone99. Cerasorb® (Curasan, Kleinost-heim, Germany) is an example of a β-TCP, having 45% to 50% porosity. The material is osteoconductive and biocompatible, but not osteoinductive.100

Straumann BoneCeramic® is a novel, fully synthetic bone substitute on the market, consisting of 60% HA and 40% β-TCP. The material is 90% porous with interconnected pores (100-500 µm in diameter).

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The TCP component is supposed to dissolve relatively quickly and the calcium and phosphate ions are suggested to stimulate bone formation. The HA component is less degradable and will protect the augmented area from resorption and subsequently maintain the volume of the graft.101 Other examples of alloplastic grafts are calcium sulphate and Bioglass with different tissue response to their resorption and dissolution.58

DentinDentin is composed of approximately 70% mineral, 20% organic matrix and 10% water by weight. The mineral part mainly consists of carbonate-substituted hydroxyapatite. The organic part contains 90% fibrous proteins (mainly type I collagen and a small percentage of type III collagen), lipids and non-collagenous matrix proteins102 similar, although not identical, with those of bone. Dentin contains BMP and possesses both osteoconductive and osteoinductive properties103-106 that might enhance bone integration compared to strictly osteoconductive bone substitutes. Demineralized dentin induces formation of new bone when implanted in muscle pouches in rats, rabbits and pigs.104, 107-109 However, allogeneic deminera-lized dentin blocks implanted in the palatal connective tissue in rats appear not to induce any bone formation up to 4 weeks after implantation.13 Furthermore, non-demineralized dentin may resorb fast with no signs of bone formation when implanted in animal muscle.104, 106, 109 Due to instability of the graft and/or ruptures in the periosteum, particulated demineralized dentin or hydroxyapa-tite ceramics applied as bone onlays are frequently encapsulated in fibrous tissue.15, 110-113 Hence, there is reason to believe that for dentin, besides the surgical technique, the demineralization procedure, and the implant environment play important roles in osteoinduction and osteoconduction.

Fibrin glueFibrin sealants are biological adhesives that have been used extensively in most surgical specialties due to the haemostatic and adhesive properties.114 The activity of the material is based on the natural clotting cascade. Fibrinogen is converted to fibrin by thrombin in the final step and cross-linking of fibrin monomers into an insoluble

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complex. When applied in contaminated sites an increased risk of infection may occur by providing a growth medium of nutrient proteins.115 Fibrin glue has been used frequently in combination with bone substitutes to make the grafting material easier to handle.16, 116,

117 However, there is a controversy in the literature whether fibrin glue has a positive or negative impact on bone formation.114

Graft healinGThe biological properties of bone grafts and bone substitutes are often described by the terms osteoinduction, osteoconduction and osteogenesis.55

Osteoconduction – is a process where biological or non-biological materials may function as a three-dimensional structure, allowing ingrowth of sprouting capillaries, perivascular tissue and osteopro-genitor cells from the recipient bed.118 This basically depends on the number and size of the channels through the graft, affecting both the rate of graft replacement and stabilization.119 The process starts within a few days after grafting.

Osteoinduction – is the process of recruitment of host mesenchymal type cells from the surrounding tissue e.g. periosteum and bone marrow, under the influence of a soluble protein called BMP.120 The BMP belongs to the family of transforming growth factors, (TGF)-β.121 The major phases of osteoinduction are: i) chemotaxis – defined as a directed migration of cells in response to a chemical gradient e.g. BMP; ii) mitosis and proliferation of newly attached mesenchymal cells; and finally iii) differentiation of the cells into chondroblasts with subsequent cartilage formation. After vascular invasion, mineralization of cartilage take place, osteoblasts are diffe-rentiated and bone formation starts,122 similar to endochondral ossi-fication.

Osteogenesis – is defined as formation of new bone from osteo-progenitor cells. Osteogenesis can be divided into spontaneous osteo-genesis, when new bone is formed by osteprogenitor cells within the wound defect, i.e. a bone fracture and transplanted osteogenesis when the formation of new bone is related to the presence of bone-forming cells within the bone-graft. In autogenous bone graft the living osteoprogenitor cells may survive and potentially differentiate to osteoblasts and eventually to osteocytes. These cells represent the osteogenic potential of the graft.123

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Healing of autogenous bone graftsThe most important factors in the mechanism of AB block integration are the amount of cellular marrow in the graft, the vascularity of the recipient site and the stability of the grafted bone achieved.124 Cells of the bone marrow, periosteum, endosteum and the outermost of bone surfaces may survive but the majority of the graft undergoes necrosis.56 Revascularization of a free bone graft may partly occur through micro anastomoses with pre-existing host vessels.125

A non-infected bone graft may be: i) incorporated in the surrounding bone and after a while sharing the same biological, mechanical och esthetical characteristics; ii) partly or totally resorbed ; or iii) sequestrated, encapsulated in fibrous tissue as a result of foreign body reaction. Incorporation is the process of envelopment and interdigitation of the donor bone or bone substitute with new bone deposited by the recipient which occurs during resorption of the old, dead grafted bone and by remodelling of the new living bone structure.120 Three phases of incorporation may be distinguished by the histologic appearance:126

Phase 1 – Inflammation (minutes to hours). The graft is surrounded by a hematoma. Platelets are attracted by the fibrin clot. Leucocytes and macrophages invade the graft after vasodilatation and exudation of plasma.

Phase 2 - Consolidation (days). Fibrovascular ingrowth proceed, osteoclasts are differentiated and starts resorption of the graft. Mesenchymal cells are recruited from the vicinity as a response to growth factors and BMPs (osteoinduction) and they differentiate into osteoblasts, which deposit osteoid on graft trabeculae, the latter serving as scaffold for the osteoblasts (osteoconduction)

Phase 3 – Remodelling (weeks-years). The graft is continuously replaced by the remodelling process from the recipient bone.The bone block graft is replaced by creeping substitution, however cortical bone may be extremely slowly replaced and parts may be unresorbed for as long as 20 years. In children, bone resorption and new tissue remodelling is much more rapid than in adults and bone grafts may not be microscopically recognizable after 2 years.40

Cortical bone hardly survives after grafting and incorporation is mainly dependent upon the tissues at the recipient site at least from a cellular point of view. In contrast, cancellous bone grafting, if performed under good conditions (e.g. minimally traumatizing

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and rapid grafting to a vascular bed without any infection) actively contribute to osteogenesis by providing large numbers of osteogenic and potentially osteogenic cells.127 In addition, the preparation of the recipient bed seems to have an important effect on bone graft integration and graft volume resistance. In an experimental study in rats, Alberius et al128 removed the cortical bone by grinding and noted that marrow exposure resulted in increased graft integration, however, some grafts became partly submerged into the recipient site. Instead, perforation of the recipient bone seemed to improve bone graft integration without submersion of the graft into the host bone.129-131

Healing of Bio-OssIn an experimental study on dogs, Araujo et al132 clarified the dynamics of Bio-Oss® collagen incorporation in the host tissue. Different phases were described; first, the biomaterial was trapped in the fibrin network of the coagulum and polymorphous nuclear (PMN) cells migrated to the surface of the particles. In a second phase the PMN cells were replaced by osteoclasts. The osteoclasts resided on OPN-positive cement lines on the surface of the particles, indicating that the cells were properly attached and apparently removed material from the surface of the xenogenic material. After 1-2 weeks the osteoclasts disappeared and were followed by osteoblasts, laying down bone mineral in the collagen bundles of the provisional matrix. The Bio-Oss® particles became integrated in bone in this third phase. Tapety et al133 investigated the response of bone cells to Bio-Oss®, which was grafted to bone defects in rat femur. After 3-5 days osteoblasts were recognized starting bone formation on the surface of the particles. TRAPase-reactive osteoclasts were not found on the trabeculae until day 7. The authors speculated that Bio-Oss® might be subjected to resorption by osteoclasts due to physiologic bone remodelling. In these studies it was assumed that proteins may cover the particles produced by different mesenchymal or hematopoietic cells132 or may have been released from the periphery of the host bone during drilling.133 The protein layer may attract the bone cells and allow them to attach to the surface of the particles. It is unknown whether the osteoclasts are able to actually resorb the deproteinized bovine bone and if the findings are

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applicable to humans. It has been suggested by some researchers that the osteoclasts presented on the graft surfaces are indeed active,88, 90,

92, 133 however, it has also been suggested that the function of the osteoclast is impaired.91 Although Bio-Oss® has excellent osteo-conductive properties, several animal studies have demonstrated delayed healing when the material has been grafted to bony defects or used with guided bone regeneration (GBR) technique.62, 134-137 For ethical reasons the process of early healing of grafting materials in humans is not possible to investigate. However, since the grafting procedure and implant insertion is often performed in 2 stages, biopsies can be harvested at the second stage without any additional surgical intervention. After sinus floor augmentation, similar tissue responses have been reported after 3 to 8 months healing periods of the graft. Inflammation and foreign body reactions are rarely present. Newly formed bone, mainly woven in character if in some areas consisting of more mature, lamellar bone, is frequently seen in direct contact with the DPBB particles. In addition the particles are often interconnected by trabecular formation. Occasionally it was possible to observe osteoclasts in close contact with DPBB particles and newly formed bone.16, 88, 138-145 In studies where AB had been added to DPBB, residual AB particles were seldom present or difficult to detect.77 In biopsies retrieved after a healing period of 3 years following sinus floor augmentation, Hallman et al77 reported that lamellar bone filled the space between the particles resulting in an overall dense appearance with the majority of the DPBB particles (>90% ) in direct contact with lamellar bone. Long-term evaluation of the fate of DPBB particles is sparse and published articles are mainly case reports or case series with a limited number of patients. After a healing period of 4.5 years,146 6 years,82 7 years,147 9 years148 and 14 years,149 histological evaluations have demonstrated the presence of DPBB particles embedded in mature lamellar bone without any inflammatory reactions. In some cases osteoclasts were evident on the surfaces of the DPBB particles and the presence of lacunae that might indicate slow resorption. It has been speculated that the slow resorption rate by osteoclasts may be caused by the high calcium concentration on the biomaterial surface. The authors also speculated that long-lasting presence of DPBB particles could be explained by a bonding mechanism that maintains the biomechanical

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integrity of the bone-biomaterial interface during the remodelling process, although not further explained in the paper.148

In a recent review on DPBB in periodontal and implant surgery it was stated that comparative studies are needed to improve evidence on clinical outcomes of DPBB used for bone augmentation procedures.76

Healing of BoneCeramic®

Ceramics do not possess any osteogenic or osteoinductive properties. The osteoconductive properties of these materials are dependent on the pore size, porosity and degradation potential. Even though the literature is not conclusive, the optimal macro pore size for tissue ingrowth seems to be between 150 µm and 500 µm.55 A definition of thresholds regarding macro pore characterization of bone substitutes has been suggested considering to part divergent literature data, namely; pore size < 60 µm for the penetration of individual cells and formation of non-mineralized tissue and pore size > 250 µm for the ingrowth of fully mineralized bone.150 Ideally, the ratio of degradation and new bone formation is 1:1, but if the degradation is faster than the bone formation, the result of the grafting procedure will end up in inadequate bone volume. The degradation in vivo may be achieved by dissolution or is cell mediated (multinuclear cells, osteoclasts and macrophages). Evidence concerning osteoclastic response to HA substrates is controversial and although osteoclasts are able to adhere to the surfaces of ceramics, this does not necessarily mean that the cells have any ability to resorb them.151-153 However, osteoclastic resorption has been demonstrated in biphasic calcium phosphate (BCP) with different HA/TCP ratios.99, 154 The cells involved and speed of resorption are determined by the local biological environment (e.g. pH, presence of cells and H2O content) and material properties (e.g. Ca/P ratio, crystallinity, particle size, surface area and porosity).97 The concept of BCP is based on the balance between the more stable phase of HA and the more soluble TCP. During its dissolution in the body, calcium and phosphate ions are released into the biological medium, which may stimulate bone formation155 or at least slow down osteoclastic activity.99, 154 Jensen at al156 evaluated the short-, mid- and long-term influence of different HA/TCP ratios on bone formation using AB and DPBB as controls

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in an experimental study in minipigs. There seemed to be a delayed healing for all bone substitutes compared to AB. Bone formation and degradation of the ceramics seemed to be inversely propor-tional to the HA/TCP ratio. This is in accordance with other animal studies.152 The amount of newly formed bone was similar for BCP 80/20, BCP 60/40 and DPBB but lower compared to AB and BCP 20/80. However, whilst in DPBB defects woven bone amalgamated and connected the particles, abundant bone formation around the BCP particles (BCP 80/20 and BCP 60/40), only near the defect walls, was the dominating feature. The degradation of BCP 80/20 and BCP 60/40 was limited, probably due to the high crystallinity.

BoneCeramic® (consisting of 60% HA and 40% TCP) has been used clinically for sinus floor augmentation18, 157 and for lateral ridge augmentation.158 In 3 clinical studies histological and histomorp-hometrical outcomes were studied comparing BoneCeramic® and Bio-Oss® 6 to 8 months after sinus floor augmentation.101, 157, 159 In all studies there was no difference in new bone formation between the materials and no inflammation or pathologic finding were reported. However, most of the bone formation took place between the BoneCeramic® particles,159 whilst the new formed bone was more intimately associated with the Bio-Oss® particles, which might reflect different osteoconductivity of the materials.101 On the other hand, the histological results of biopsies retrieved 5 months after sinus floor augmentation revealed god tissue integration and direct bone to particle contact in both sites augmented with BoneCeramic® and

Bio-Oss®. 143 In the same study there was significantly more residual bone substitute material in the Bio-Oss® group but no difference in new bone formation between the groups. In all studies it was concluded that there is a need for further investigations regarding the clinical relevance of the properties of BoneCeramic®.

surGical techniquesSinus floor augmentationDue to resorption of the alveolar crest following tooth extraction in combination with pneumatization of the maxillary sinus, there is often lack of adequate bone volume for implant treatment and a need for bone grafting in the posterior maxilla. Sinus floor augmen-tation was first introduced by Boyne and James,160 who described

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a surgical technique, where the sinus floor was grafted with parti-culated cancellous bone from the iliac crest after the lateral sinus wall had been fenestrated and the Schneiderian membrane had been elevated. The procedure has been frequently used since, with excellent clinical results.74 Due to the drawbacks of AB grafting, several different bone substitutes have been used alone or in combination with AB. A consensus on the most appropriate material in sinus floor augmentation has not been reached despite several systematic reviews and meta-analyses have been conducted on the topic.21, 23, 74,

161 The most frequently used bone substitutes include demineralized freeze-dried bone, hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), deproteinized bovine bone (DPBB) and combinations of these materials.23 The percentage of total bone volume (TBV) in a histologic section of the graft has been a frequently used parameter to assess the performance of a bone graft or bone substitute in an augmented area.17, 161-163 Recently two meta-analyses were performed to analyse the TBV after sinus floor augmentation with different grafting materials.23, 161 Handschel et al23 reported that AB shows the highest TBV values in a relatively early phase after grafting, i.e. 4 to 9 months, but after 9 months there were no significant difference between the different grafting materials. Additionally, only DPBB, β-TCP and AB (and the combinations of AB and these materials) presented evaluable data for meta-analyses. In contrast, Klijn et al161 reported that TBV was significantly higher before 4.5 months and after 9 months of AB graft healing compared to the period in between. For all other bone substitutes no significant effect on TBV in time could be proven. The authors stated that AB still has to be considered to be the gold standard. However, in an interven-tion review to determine which are the most effective augmentation procedures of the maxillary sinus, it was concluded that Bio-Oss® and Ceracorb® (Curasan AG, Kleinostheim, Germany) (β-TCP) appear to be as effective as AB and can be used as replacement for AB grafting.22 Furthermore, it has yet to be proven if TBV is an adequate variable to evaluate the clinical outcome of graft and implant success.

Lundgren et al164 described a sinus membrane elevation technique using a lateral approach with a replaceable bone window, showing promising long-term results.165 After solely sinus membrane elevation

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and simultaneous implant installation, a space filled with blood is created allowing for bone formation according the principles of tissue generation,166 without the use of any filling material.165 The sinus becomes a self-contained defect, promoting space maintenance and coagulum stability, allowing continuous blood supply from the bony wall and cell migration, resulting in favourable healing conditions.139 Although the surgical technique seems demanding and operator sensitive, the reduced morbidity and lower costs for the patient makes the procedure a highly interesting alternative to using grafting materials. Apart from TBV, the amount of newly formed bone is also considered an accurate indicator to assess and compare the healing potential of graft or bone replacement graft materials after sinus floor augmentation.159 According to the results of Lundgren et al, it could be questioned whether sinus floor augmen-tation is a suitable environment or not to test the osteoinductive and osteoconductive properties of different grafting materials per se. Furthermore, when future studies are conducted to test the performance of grafting materials the described technique should be considered used as control.

Lateral ridge augmentationLateral ridge augmentation procedures are necessary when the width of the recipient alveolar crest presents with inadequate dimensions for implant placement. A number of surgical procedures have been performed to create adequate bone width either in combination with implant placement (associated with dehiscenses or fenestration bone defects) or following a period of graft healing.167 These procedures include guided bone regeneration (GBR) alone or in combination with grafting materials, osteodistraction, “split-ridge” osteotomy for lateral expansion and bone grafting with different grafting materials (AB, allograft, xenograft and alloplastic materials). The grafting materials may be used as blocks (onlay) or particulates. In recent reviews, it was not possible to demonstrate the superiority of one augmentation technique over another.43, 66

The principal feature of GBR is to create a secluded space to allow presumably slower moving osteoprecursor cells to migrate from the surrounding bone rims and simultaneously prevent cellular ingrowth from the surrounding soft tissue, so that the osteoge-

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nesis may occur unhindered. This situation is provided by a semi-permeable membrane allowing free diffusion of tissue-fluids and nutrients.53 In addition, to excluding unwanted cells, the membrane also acts to stabilize the blood clot.168 The technique was first described by Dahlin et al,166, 169 who tested the ability of generating new bone around titanium implants with GBR in a rabbit model. GBR is frequently combined with a filler material (i.e. AB or a bone substitute) to support the membrane, stabilize the blood clot and reduce its volume and shrinkage.170 The membranes can be resorbable or non-resorbable. If a particulated graft is used, a membrane to cover the graft is advocated.171 One common complication in GBR is soft tissue dehiscenses and exposure of the membrane which might lead to bacterial infection and compromised graft healing, especially in non-resorbable membrane sites. In addition, another disadvan-tage with non-resorbable membranes is that it must be removed in a second surgical intervention.

In larger edentulous defects, autogenous bone used as onlay grafts have been advocated,172 however; the morbidity from the donor site and the higher costs compared to for example GBR 173 in combination with filling material, must be considered in the choice of surgical procedure to be utilized. Lateral augmentation with Bio-Oss® has been performed in both animal studies174-176 and human studies.79, 80,

177 It appears that Bio-Oss® can be used with a slightly higher risk of having an implant failure.44 Since Bio-Oss® only possesses ostecon-ductive properties, AB has been added to DPBB in different ratios, in grafting procedures, to add osteoinductive properties and subse-quently enhance bone formation.22, 77, 79, 80, 90, 173, 178 However, the optimal ratio of Bio-Oss® and AB, and its effect on bone formation is not known.63 To the best of the author´s knowledge, different ratios of Bio-Oss® and AB have not been compared for lateral augmenta-tion in a clinical, randomized and controlled study.

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aiMs

General aimThe overall aim was to clinically and histologically study the short- and long-term tissue reactions to and resorption of bone substitutes after bone augmentation.

specific aims

1. To examine the influence of partial demineralization of xenogenous dentin on bone formation in an osteoconductive environment (paper I).

2. To evaluate the tissue response to BCP and DPBB after 3 years of healing and to evaluate the implants placed in the grafting materials after sinus floor augmentation (paper II).

3. To study the long-term clinical outcome of dental implants and graft reduction in maxillary sinuses grafted with DPBB (paper III).

4. To histologically and histomorphometrically evaluate the long-term tissue response and possible resorption of DPBB particles grafted to the maxillary sinus (paper IV).

5. To study the radiological changes and healing of DPBB with different percentages of AB used for lateral augmentation of the alveolar crest (paper V).

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hypOtheses

• Partial demineralization of xenogenous dentin has an influence on bone formation in an osteoconductive environment (paper I)

• There is no difference in clinical outcome between implants placed in BCP or DPBB after 3 years of functional loading (paper II)

• There is no difference in clinical outcome between implants placed in a mixture of 80% DPBB and 20% AB or residual bone after 10 years of functional loading (paper III).

• There is no long-term resorption of DPBB particles after maxillary sinus floor augmentation (paper IV)

• There are no histomorphometric or other differences in width- and volumetric reduction of two different mixtures of DPBB and AB used for lateral ridge augmentation (paper V)

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Materials and MethOds

subjects and experiMental OutlinesPaper IThe study was conducted to examine the influence of partial demine-ralization of xenogenous dentin on bone formation in an osteocon-ductive environment. Forty adult rats with a body weight ranging from 340 to 400 g were used. Dentin blocks with different demi-neralization time were placed subperiostally as onlay grafts on the cranial bone on the one side and as control a non-demineralized dentin block on the other side or the site was left empty creating the following 8 groups with 5 rats in each group:

table 1. Data of the different subgroups in paper I

Group n experimental site control site

1 5 1 h ND

2 5 1 h SD

3 5 2 h ND

4 5 2 h SD

5 5 6 h ND

6 5 6 h SD

7 5 12 h ND

8 5 12 h SD

h = hours of demineralizationND = Non-demineralized dentin blockSD = Only subperiosteal dissection – no dentin block

The results were assessed after four weeks by routine histology.

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Paper IIThe purpose of this 3-year follow up of a randomized and controlled study was to evaluate and compare the clinical and histological outcome of BCP and DPBB used for sinus floor augmentation and implants placed in the grafting materials.

Eleven patients (6 females and 5 males) with a mean age of 67 years, range (50-79 years) consecutively referred to the Department of Oral & Maxillofacial Surgery, Gävle county hospital, in need of bilateral sinus floor augmentation prior to implant treatment were included in this study. Nine patients were edentulous and 2 patients were partially edentulous. The patients received BCP (test) in one maxillary sinus and DPBB (control) on the contralateral side after a randomization procedure. After a graft healing period of 8 months, 62 dental implants (SLActive, Straumann®, Basel, Switzerland) were installed. The patients received their fixed prosthesis after an implant healing period of 4 months (mean 3.9, range 3.2-7 months). The patients have been followed up at base-line, at 1-year and at 3-years of functional loading. In this 3-year follow-up clinical examination, radiographic examination and resonance frequency analyses (RFA) were performed. Implant survival and success rates were calculated. A modification of the criteria for implant success described by Albrektsson et al179 was used (marginal bone loss not exceeding 2 mm after 1 year and 0.2 mm every subsequent year). In addition 18 biopsies were retrieved from the augmented sinuses (9 from DPBB and 9 from BCP) for qualitative histological evaluation and histomorphometry.

Paper III and IVThe aims of these studies were to evaluate the long-term success of dental implants and graft reduction after sinus floor augmentation with a mixture of DPBB and AB (paper III) and to histologically and histomorphometrically evaluate the long-term tissue response to and possible resorption of DPBB particles (paper IV).

In 20 consecutive patients (14 women and 6 men) with a mean age of 62 years (range 48-69 years) 30 maxillary sinuses were augmented with a mixture of 80% DPBB and 20% AB. After a healing period of approximately 6 months biopsies were retrieved from 16 of the 20 patients and 108 dental implants were placed. Clinical results,

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including marginal bone levels, have previously been evaluated after 1, 3 and 5 years of functional loading. After 10 years of functional loading 14 of the 20 patients were clinically evaluated and reduction of the grafts were radiologically calculated (paper III). Eleven of the patients (of which 9 could be compared with biopsies retrieved 6 months after grafting) volunteered to additional biopsy retrieval from the grafted area (paper IV) and histological and histomorp-hometrical evaluations were performed. The DPBB particle sizes were compared with samples harvested from the same patients at 6 months and pristine particles from the manufacturer.

Paper VThe aim of the study was to radiologically and histologically evaluate the graft healing and volumetric changes after lateral ridge augmen-tation with 2 different compositions of DPBB and AB.

Included in this study were thirteen patients with 14 edentulous or bilaterally partially edentulous jaws (6 males and 7 females,) with a mean age of 59.6 years (range; 29-75) consecutively referred to the Department of Oral & Maxillofacial Surgery, Gävle county hospital, and in need of lateral augmentation of the alveolar crest prior to implant treatment.

After randomization, lateral augmentation with a mixture of 60% DPBB and 40% AB was performed on the one side and a mixture of 90% DPBB and 10% AB was performed on the cont-ralateral side. All grafts were covered by collagenous membranes. Cone beam computed tomography (CB/CT) was obtained preope-ratively, immediately postoperatively and after a healing period of 7.5 months. After a healing period of 8.1 months (range; 7.9-8.3) biopsies were retrieved from each graft and dental implants were installed. Width and volume changes were measured on CB/CT scans. In addition, histomorphometry and a histological evaluation of the biopsies were performed.

ethical cOnsideratiOnsStudy I was approved by the local ethics committee on animal experiments, Stockholm, Sweden and studies II-V were approved by the regional ethical review board in Uppsala, Sweden. All patients gave their informed consent.

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preOperatiVe exaMinatiOns, inclusiOn and exclusiOn criteriaPrior to surgery all patients were clinically and radiographically examined. The radiographic examinations were based on panoramic radiographs (papers II-IV), conventional tomography (Scanora®, Soredex orion, Corporation Ltd, Helsinki, Finland) (papers II-IV) and cone beam computed tomography (i-CAT® Cone Beam 3-D Imaging System, Imaging Sciences International, Inc., Hatfield, PA, USA) (papers III, IV and V). The residual bone was examined with respect to width, height, regional anatomy and pathologic conditions.

Included in the studies were consecutive patients with a residual bone of the maxillary sinus floor being less than 5 mm, bilaterally (paper II) or unilaterally/bilaterally (papers III and IV). In paper V, totally or partially, bilaterally edentulous consecutive patients with adequate bone height but bone width ≤ 4 mm were included in the study. Patients were excluded if they had any uncontrolled systemic disease, history of radiation to the area of treatment, uncontrolled periodontal disease, active sinus infection (papers II-IV) or any smoking habits (paper V). In paper II-IV patients were not allowed to smoke more than 10 cigarettes and/or were advised to refrain from or reduce their smoking habits.

GraftinG MaterialsDentinIn paper I sixty dentin blocks, 2-3 mm thick and 4 mm in diameter, were prepared with a trephine bur from unerupted developing pig teeth from which the enamel/enamel matrix had been removed. Forty of the dentin blocks were demineralized in 24% EDTA (pH 7.0) for 1 hour, 2 hours, 6 hours or 12 hours, respectively in room temperature. Another twenty dentin blocks were prepared but not demineralized. After thorough rinsing in tap water, the blocks were stored for one to two months in a freezer at -18°C before implantation.

Biphasic calcium phosphate (BCP)In paper II, 100% of BoneCeramic® (Institut Straumann AG, Basel, Switzerland) mixed with saline was used to augment one side of the maxillary sinuses. BoneCeramic® is a fully synthetic, biphasic bone substitute which consists of 60% HA and 40% β-TCP in a hard

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sintered mixture with a crystallinity of 100%. BoneCeramic® offers an interconnected porosity of 90% with pores ranging from 100 to 500 µm and a particle size that varies between 0.25 and 1 mm.

Deproteinized bovine bone (DPBB)BioOss® is a natural bone mineral made from bovine bone (extre-mities obtained from Australian cattle claimed to be prion-free) via a proprietary extraction procedures. The organic material has been extracted by prolonged heat treatment (15 hours in 300°C) and several chemical purification processes, including strong alkaline treatment over a period of several hours. The final product is γ-irradiated with at least 25 kGy. BioOss® exhibits a porosity (70-75%) and crystalline structure similar to human bone96. The particle size used in the present studies was 0.25-1 mm.

In paper II, 100% of BioOss®, mixed with saline was used to augment one side of the maxillary sinuses. In papers III and IV, 80% BioOss® was mixed with 20% AB, blood and fibrin glue before augmenting the maxillary sinuses. In paper V, 2 mixtures were used for lateral ridge augmentation, 90% BioOss® and 10% AB or 60% BioOss® and 40% AB. Both compositions were mixed with autogenous blood and fibrin glue before grafting.

Autogenous boneIn papers III and IV AB grafts were retrieved from the chin region and in paper V AB grafts were retrieved from the retromolar area. All bone grafts were particulated in a bone mill.

Fibrin glueIn papers III, IV and V, 0.5 ml of fibrin glue (Tisseel®, Duo Quick Immuno, Vienna, Austria) was added to each graft in order to make the graft mouldable and avoid the particles to migrate from the augmented area. The material is a two-component fibrin sealant. The sealer protein solution contains human fibrinogen, fibronectin, plasminogen, factor XIII, albumin and a synthetic fibrinolysis inhibitor, aprotinin, which helps prevent premature degradation of the fibrin clot. The thrombin solution contains human thrombin and calcium chloride. When mixed together, the 2 components combine and mimic the final stages of the body’s natural clotting cascade to form a rubber-like mass.

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MembraneBio-Gide® (Geistlich Pharmaceutical, Wollhausen, Switzerland) is a bilayer, resorbable non cross-linked collagen membrane of porcine origin. The smooth upper layer is supposed to lead to favourable healing of soft tissue. The dense porous layer may act as a guide for bone forming cells. Bio-Gide® was used in paper II to cover the lateral window after sinus floor augmentation and in paper V to cover the grafts after lateral ridge augmentation. The purpose of the membrane was to prevent early soft tissue ingrowth, stabilize the grafted area and blood cloth and subsequently enhance bone formation.

dental iMplantsIn paper II, 62 Straumann® SLActive implants were installed and followed for 3 years of loading. Twenty-four implants were placed in sites grafted with BCP, 23 implants in sites grafted with DPBB and 15 implants were placed in residual bone. The implants were 8-12 mm in length and 3.3 or 4.1 mm in diameter.

In paper III, 108 commercially pure titanium (c.p) implants with a turned (machined) surface (Mark II, Brånemark System®, Nobel Biocare AB, Gothenburg, Sweden), 7 to 18 mm in length and 3.75 mm in diameter, were installed. Seventy-nine implants were inserted in grafted bone and 29 in residual bone. At 10-years follow-up, 68 implants (53 implants placed in grafted maxillary sinuses and 15 implants placed in residual bone) were evaluated.

drOp-OutsPaper I - two dentin blocks in the non-demineralized control group were infected or dislocated and were excluded from the analyses.

Paper II - two patients had cemented restorations and were excluded from the evaluation regarding RFA and they did not undergo CB/CT. Two patients refused to participate in biopsy retrieval. Eight out of the 18 retrieved biopsies did not contain any representative grafting material due to difficulties in biopsy harvesting and were excluded from the analyses; subsequently 10 biopsies were available for paired analyses (5 in BCP and 5 in DPBB).

Paper III and IV - there were in total 6 patient dropouts at 10-years follow-up. Three patients were deceased, two were too ill

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to participate and one had moved, representing 25 unaccounted for implants. Another implant was “sleeping” and was not included in the 10-years follow-up (paper III). Three patients did not want to participate in the biopsy retrieval. Out of the 11 biopsies retrieved, 9 could be matched with the patients who had biopsy retrieval at 6 months (paper IV).

Paper V - there were no drop-out patients and all biopsies were available for histomorphometry in this study.

surGical prOceduresAnimal study (paper I)With the rats under general anaesthesia (intramuscular injection of fentanyl citrate (0.4 ml equivalent to 0.126 mg/kg) body weight, HypnormTM; Janssen Pharmaceutica, Beerse, Belgium), a paramedian incision was made from the occipital to the frontal region. A pocket was created on each side of the midline after subperiosteal dissection. One demineralized dentin block was placed on one side and on the other side a non-demineralized block was placed or the pocket was left empty. No further fixation was used. The wounds were closed with interrupted non-resorbable sutures.

Sinus floor augmentation (Papers II-IV)As a prophylactic measure, the patients received 1 g of penicillin-V (Kåvepenin, Astra AB, Södertälje, Sweden) three times daily for 7 days (paper II) or 2 g penicillin-V and 500 mg of metronidazole (Fasigyn, Pfeizer, Stockholm, Sweden) preoperatively and 2 times daily for 10 days (papers III and IV).

The sinus floor area was prepared under local anaesthesia as described elsewhere.16, 160 In brief; a crestal incision with vertical releasing incisions was performed, a mucoperiosteal flap was raised and the lateral wall of the sinus was exposed. An approximately 2 cm x 1 cm big window was outlined with a round bur. The Schneiderian membrane and the attached bone in the centre of the window were elevated after careful subperiosteal dissection (Figure 1). Although special care was taken, the Schneiderian membrane was accidentally perforated during the elevation procedures in 6 out of 22 sinuses in paper II and in 9 out of 30 sinuses in papers III and IV.

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In paper II, 100% BCP (mean 1.0 g, range 0.5-1.5 g) was augmented in one maxillary sinus floor and 100% DPBB (mean 0.8 g, range 0.5-1.0 g) on the contralateral side according to a randomi-zation procedure. A collagen membrane (Bio-Gide®) was placed on the lateral windows to prevent soft tissue ingrowth.

In paper III and IV, DPBB was mixed with autogenous bone particles in an 80:20 mixture (measured by weight), and fresh, autogenous blood from the wounds. Tisseel® was added to increase the malleability of the graft material and to avoid the particles to migrate and accidently penetrate perforations in the sinus membrane. The graft mixture was packed layer by layer and thrombin was added to catalyse the setting of the fibrin glue. Resorbable sutures were used to close the wounds.

Lateral ridge augmentation (Paper V)All procedures were performed by the same surgeon (AM) under oral sedation with 5-10 mg midazolam and local anaesthesia (Dormicum, Roche AB, Stockholm, Sweden).

figure 1. Sinus floor elevation. The window has been outlined with a round bur (A) and the sinus membrane was carefully elevated (B).

As a prophylactic measure all patients received 2 g of V-penicillin (Kåvepenin, Astra AB, Södertälje) x 3 for 10 days starting 1 hour preoperatively or clindamycin (Dalacin, Pfizer AB, Stockholm, Sweden) 300 mg x 3 for 10 days in event of penicillin allergy. Anaesthesia was induced by lidocaine (2%) with epinephrine (1:80,000) (Xyloxcain/adrenalin, Astra AB). After a slightly lingual oriented crestal incision and vertical releasing incisions, a mucope-riosteal flap was elevated bilaterally and reflected buccally. At the

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edentulous areas the vertical incisions were at least 5 mm beyond the alveolar crest to be augmented and in dentate areas at least one tooth away from the surgical site. Care was taken to free the buccal wall from periosteum and the cortical bone was perforated with a small round bur (Figure 2A).

Through a careful incision of the periosteum from one releasing incision to the other, the mucoperiosteal flap was elongated to achieve a passive closure of the incision. The grafts were placed on the buccal aspect of the alveolar ridge (Figure 2B), layer by layer, gently pressed to the recipient site. Thrombin was added to catalyse the setting of the fibrin glue (Figure 2C). Collagen membranes were used to cover all grafts (BioGide®) without any further fixation (Figure 2D). The incisions were closed with horizontal mattress, interrupted and continuous resorbable sutures (4-0 Vicryl®, Johnson & Johnson AB, Sollentuna, Sweden.

Autogenous bone harvestingHarvesting of AB was performed under local anaesthesia and sedation with either (papers III and IV) diazepam (10-25 mg, per oral Apozepam, Dumex-Alpharma AB, Stockholm, Sweden) or

figure 2 (a-d). Edentulous mandible after exposure and perforation of the buccal bone (A) augmentation with the graft mixture (B) and addition of thrombin (C) and membranes (D).

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midazolam (5-10 mg, Dormicum, Roche AB, Stockholm, Sweden) (paper V). Anaesthesia was induced by lidocain (2%) with epinephrine (1:80,000) (Xylocain /adrenalin, Astra AB, Södertälje, Sweden).

In paper III and IV (chin grafts), the mandibular symphysis was exposed through a mucosal and musculoperiosteal incision from canine to canine in the deepest part of the vestibule and the lip. A unicortical osteotomy was performed by the means of a thin fissure bur. The osteotomies were prepared at least 4 mm superior to the mandibular inferior border and 5 mm inferior to the root tips. The bone graft was harvested with a thin osteotome. Particulation of the bone graft was performed using a surgical bone mill (Tessiers Osseous Microtome; Mühlheim-Stetten, Germany). The periosteum, the muscle attachment and the mucosa were carefully sutured in layers using resorbable sutures.

In paper V, the bone graft was retrieved from the retromolar area. An incision was performed 5 mm inferior to the attached gingiva from the first premolar to the anterior part of the mandibular ramus. A monocortical AB block of approximately 2-3 cm x 1 cm was retrieved by means of a thin fissure bur and a bone mill (Roswitha Quétin Dental Produkte, Leimen, Germany) was used to particulate the bone block. The harvesting of the bone graft was performed unilaterally in all cases. The particulated bone was mixed with DPBB in different ratios by weight using a digital scale.

Healing time of grafts and implantsPaper I - after a healing period of 4 weeks the rats were sacrificed and the specimen were fixed in 4% neutral buffered formalin.

Paper II - after a mean graft healing time of 7.7 months (range; 7 to 8.3), dental implants were placed non-submerged and were left to heal for an average healing time of 3.9 months (range; 3.2 to 7 months) before loading.

Paper III and IV - after a mean graft healing period of 6.7 months, submerged dental implants were placed. The implants were exposed and healing abutments were connected after an implant healing period of 6-8 months (mean; 6.7 months).

Paper V - dental implants were placed on average 8.1 months (range; 7.9 to 8.3 months) after the grafting procedure.

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Biopsy retrieval (papers II, IV and V)Core samples were harvested perpendicular to the lateral wall of the augmented maxillary sinus by the means of a trephine bur (3-4 mm in diameter), 6-8 months; mean 6.7 months (paper IV), 3 years (range; 35 to 37 months) (paper II), and 11 years; mean 11.5 years (paper IV) (Figure 3) after the grafting procedure. In paper II, 18 biopsies were retrieved (9 from BCP and 9 from DPBB). In paper IV, 11 biopsies were retrieved after 11 years, of which 9 were from the same patients who had biopsies retrieved after 6-8 months of graft healing.

figure 4. Eight months after lateral ridge augmentation, Astra osseospeed implants were

placed and biopsies were retrieved from both DPBB:AB compositions perpendicular to

the alveolar ridge 5-7 mm inferior to the top of the alveolar crest (black arrows).

figure 3. Biopsy retrieval, perpendicular to the lateral bone, 11 years after sinus floor augmentation was performed with a trephine bur (3.1-4 mm in diameter).

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In addition, non-treated DPBB particles from the manufacturer were packed in a roll of rice paper and treated as the other samples. In paper V, after a graft healing period of mean 8.1 months (range 7.9 to 8.3 months), at the time of implant placement, a trephine bur (3.1 mm in diameter) was used to retrieve 28 biopsies (14 from the 90:10 mixture and 14 from the 60:40 mixture) in a horizontal direction 5-7 mm from the top of the alveolar crest and at least 10 mm from the midline in edentulous cases (Figure 4). All biopsies were immediately immersed in formalin fixative and sent to the laboratory for further processing.

clinical fOllOw upClinical follow-up of dental implants was performed after 3 years of functional loading, after sinus floor augmentation with 100% BioOss® or 100% BoneCeramic® (Paper II) and after 10 years of functional loading, after sinus floor augmentation with a mixture of 80% BioOss® and 20% autogenous bone (paper III).

The following clinical variables were registered in paper III: pocket depths (PD) and bleeding on probing (BoP). In paper II, plaque index (PI) and sulcus bleeding index (SBI) was added.

Resonance frequency analyses (RFA) of the implants were performed by means of the Osstell MentorTM instrument using a SmartPeg (SmartPeg Type II, Integration Diagnostics AB, Göteborg, Sweden) after 3 years (paper II) and 10 years (paper III) of functional loading. After removal of the abutment, the SmartPeg was manually inserted in the implant body. Data collected were expressed as Implant Stability Quotient (ISQ). An ISQ value between 1 and 100 is given where 1 is the lowest degree of stability and 100 the highest. The mean of 4 measurements per implant, 2 from the buccal side and 2 perpendicular to the first measurements (paper III) or 2 measure-ments (paper II), was used as the final ISQ value.

Radiographic examinationsMarginal bone level/loss (papers II and III)Intra oral radiographs were obtained for examination of the marginal bone level at baseline (bridge delivery in paper II and abutment connection in paper III), 1, 3 (paper II), 5 and 10 years (paper III) of functional loading. In paper III all but the 10-year radiographic examinations were performed with a Philips Oralix 65 apparatus

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(Philips, Milano, Italy) using Kodak Ektaspeed PlusTM dental film (Eastman Kodak). The intra oral radiographs at 10-years follow-up in paper III and 3-years follow up in paper II, were performed with Schick CDR wireless system, Schick Technologies, Inc. (Long Island City, NY, USA). Marginal bone measurements of the implants were made twice (with a 6-months interval between measurements) at the mesial and distal aspects from the reference point of the implants.

Graft dimension changes (papers III and V)Paper III - the radiographic examinations at 3 months, 1 year and 2 years after sinus floor augmentation included panoramic radiographs and cross-sectional tomograms (Scanora®). The measu-rements of the grafted area were estimated with a special ruler directly on the radiographs, taking the magnification into considera-tion (panoramic film x 1.3, tomograms x 1.7). The maximal length, height and width of the grafts were marked with a soft pen and measured to the nearest 0.5 mm. The height and length of the grafts were measured on the panoramic film and the height and width of the grafts were measured on the Scanora tomograms. A mean of the height measurements from the two techniques were calculated.

Cone beam computed tomography (CB/CT) (i-CAT® Cone Beam 3-D Imaging System, Imaging Sciences International, Inc, Hatfield, PA, USA) was obtained at 10-years follow-up after removal of the prosthetic constructions. The maximal length, height and width of the grafts were measured on the panoramic- and axial views using the software in i-CAT® Vision. To test the repeatability of the measu-rements, the radiographs of 10 randomly selected patients were measured twice with a 6-months interval between measurements.

Paper V - Cone beam computed tomography (i-CAT® Cone Beam 3-D Imaging System, Imaging Sciences International, Inc, Hatfield, PA, USA) (CB/CT) scans were obtained preoperatively, immediately after grafting and 7.5 months after the grafting procedure. Volumetric measurements were performed from all CB/CT scans in the ImageJ (version 1.45, downloaded from http://rsb.info.nih.gov/ij/download.html). Each 0.4 mm or 0.3 mm slice was displayed on a computer monitor and a drawing function was used to manually outline the margins of the graft in order to calculate the graft area for each coronal slice. The graft volumes were estimated according to the method described by Uchida,180 using the following formula:

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nV = ∑ dS x ∆hi =1

The volume of each section was dV = dS x ∆h where dS is the area of the graft in a given section and ∆h is the slice thickness of the section. Hence, the volume (V) of the graft to a height of n mm was calculated as the sum of the volumes of each section (dV).

All width measurements were made on sagittal slices perpendi-cular to the longitudinal axis of the alveolar crest, using i-Cat vision software, 3 mm and 6 mm from the top of the crest, at the anticipated implant sites. In the midline of the jaws, on the coronal view, an anatomic reproducible landmark (e.g. the nasal spine or foramen incisivum) was defined and a straight line was drawn through it. The distance from the measuring point to this line, at the middle of the alveolar crest, was obtained using the software ruler. The same anatomic landmarks and distances were used for measurements on CB/CT scans immediately after grafting and 7.5 months after the grafting procedure (Figure 5). The width of the residual bone and the total width (residual bone + bone graft) after grafting were obtained from the CB/CT scans immediately after grafting, using the software ruler. On the CB/CT scans 7.5 months after grafting only the total width was measured at the anticipated implant sites. The graft width after 7.5 months was calculated by subtraction of the total width with the width of the residual bone obtained from the scans immediately after grafting (Figure 6).

Health of the maxillary sinusesThe CB/CT scans were used to examine the health of the maxillary sinuses. In paper II, the individual maxillary sinus was classified as unhealthy if there were any mucosal hypertrophy or fluid present. In addition, in paper III, the hypertrophy of the Schneiderian membrane was divided into two groups; 1-6 mm and > 6mm.

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figure 5. Axial view of the CB/CT scans immediately after grafting (A) and 7.5 months of graft healing (B).

figure 6. (A) The total width (white arrow) and the width of the residual bone (black arrow) were measured on CB/CT scans immediately after grafting. (B) Total width 7.5 months after grafting.

histOlOGy and histOMOrphOMetryPaper I - the specimens were demineralized in neutral buffered 24% EDTA, dehydrated and embedded in paraffin. Serial sections of 7 µm thick slices were cut transversally through the midpart of the xenogenous dentin onlays, stained with haematoxylin and eosin and analysed in a light microscope. All xenogenous dentin onlays and recipient sites were evaluated semi-quantatively as follows:

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• Inflammation; none (0), slight (1), moderate (2) and intense (3). • Encapsulation; (% of the implant surface); < 50% (0) and

> 50% (1).• Resorption; (% of the implant surface); < 50% (0) and > 50% (1).• Bone formation; (% of the implant surface); 0% (0), 0-33% (1),

33-67% (2) and 67-100% (3).

Paper II, IV and V - the specimens were immersed in 4% neutral buffered formaldehyde solution, rinsed in water followed by dehydration in a graded series of ethanol, pre-infiltrated in diluted resin and embedded in light-curing resin (Technovite® 7200 VLC, Kulzer & Co GmbH, Wehrheim, Germany). The samples were divided in the long axis of the biopsy and mounted on a supporting plexi-glass slide. One thick section was taken from the centre of each biopsy and ground to a thickness of approximately 10-15 µm, according to the technique described by Donath and Breuner 181 using the Exakt® system (Exakt Apparatebau, Norderstedt, Germany) and stained with toluidine blue mixed with 1% pyronin-G. The undecal-cified, histologically stained sections were analysed qualitatively and quantitatively in a Nikon Eclipse 801 light microscope (Belmont, CA, USA) (paper II) and/or a Leitz Aristoplan light microscope (papers II, IV and V). The latter was equipped with a Microvid unit, i.e. a semi-automated image analysis system (Ernst Leitz GmbH, Wetzlar, Germany) enabling ”direct measurements” using a mouse connected to the computer. All quantifications were performed directly in the eye-piece of the microscope. In order to standardize the histomorp-hometry a region of interest (ROI, 6.67 mm2 in paper II and 3.3 mm2 in paper IV) was chosen in a representative area of the biopsy where grafting material was present, using a square grid. In paper V the 2.5x objective was used, resulting in a 25x magnification, revealing the entire biopsy in the field of view for quantitative analyses. The same person performed all the measurements in paper IV and V and another person performed all the measurements in paper II.

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The following histomorphometrical variables were measured:

table 2. Histomorphometrical measurements performed in study II, IV and V.

paper ii iV V

Total length of the graft in mm X

Ingrowth of newly formed bone into the graft (%) X

Bone, area in % X X X

Bone, area in % facing residual bone and periosteum X

Graft, area in % X X X

Graft, area in % facing residual bone and periosteum X

Soft tissue (ST), area in % X X X

ST, in % facing residual bone and periosteum X

Degree of graft material to bone contact (%) X X X

Length and area of graft particles (mm and mm2) X

Number of particles in ROI or graft X X

statisticsPaper I Firstly, for each of the four outcome variables (inflammation, encap-sulation, resorption and bone formation) the Wilcoxon signed-rank test was performed to determine presence of differences between control (non-demineralized dentin) and demineralization regardless of time (1 hour, 2 hours, 6 hours, or 12 hours of demineralization). The Bonferroni-Holm method was used to compensate for possible mass significance. Secondly, for each of the four outcome variables (inflammation, encapsulation, resorption and bone formation) a Kruskal-Wallis test was performed to determine presence of differences between the times of demineralization. The Bonferroni-Holm method was again used to compensate for possible mass significance. Adjusted p-values equal to or smaller than 0.10 were considered statistically significant. If the tests showed any statistical significant differences, the next step was to perform pair wise comparisons between 1 hour and 2 hours, 6 hours, and 12 hours of demineralization. The Wilcoxon rank sum test was used for the pair wise comparisons.

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Paper IIOne-way ANOVA test was performed to determine differences in RFA, SBI, PI, BoP, pocket depth, marginal bone level and survival/success rate between implants placed in BCP, DPBB or residual bone after 3 years. Pair wise comparisons to determine presence of differences between the groups were performed using paired t-test. To determine presence of differences in marginal bone loss from base-line to 3-years follow up for implants placed in residual bone, BCP and DBB, respectively, paired t-test was performed. The Wilcoxon signed rank test was used to determine presence of histomorphometrical differences between BCP and DPBB.

Paper IIIResults were reported as mean values and standard deviations. The individual implant, of which there were several within each patient, was the experimental unit in the analyses. A model involving generalized estimating equations (GEE) was used for handling the repeated measures on patients in the analyses of effects of residual bone/graft and smoking habits on mean marginal bone level, mean pocket depth, and RFA at the 10-year follow up. The model was set up with the main factors; residual bone/graft, smoking habits and time with the interaction term time x smoking habits to analyse changes in marginal bone level with time. The Sidak correction method was used to adjust all p-values for multiple comparisons. To analyse the differences with time in graft length, height and width, ANOVA for repeated measures was used. The chi-square test was used to analyse differences in BoP between implants placed in grafted bone and residual bone. In papers II-V a statistically significant difference was considered at p < .05.

Paper IVThe mean length and area of the particles were estimated for each patient and time-point. Wilcoxon signed-rank test was used to analyse differences in particle lengths and areas between 6 months and 11 years. The Mann–Whitney test was used to analyse differences in the particle lengths and areas between 11 years and pristine particles from the manufacture. The tests were two-tailed. Results were reported as mean values and standard deviations.

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Paper VHistomorphometric and volumetric differences between 90:10 and 60:40 (DPBB:AB compositions) were tested using Wilcoxon signed-rank test, and non-parametric confidence intervals was estimated using bias-corrected and accelerated (BCa) bootstrap-ping. Differences in width changes between the two compositions, adjusted for initial graft width were tested using mixed models, with DPBB:AB composition as a fixed factor and jaw as random factor. Model assumptions, i.e. normally distributed random effects with constant variance, equal across compositions, was verified by visual inspection of graphs of residuals and predicted values, in combination with Shapiro-Wilks test of normality. Results were reported as mean values and standard deviations.

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results

clinical resultsPaper IIAfter 3 years of functional loading, one implant placed in BCP and one implant placed in DPBB were lost resulting in an overall implant survival rate of 96.8% (BCP 95.8%, DPBB 95.7%, and residual bone 100%). There were no significant differences between the groups regarding implant survival, implant success (BCP 91.7%, DPBB 95.7%, residual bone 86.7%), PI and SBI (BCP 38.7%, DPBB 37.1%, residual bone 24.2%), BoP (BCP 38.3%, DPBB 36.7%, residual bone 25%) and pocket depth (BCP 2.6 ± 0.8 mm, DPBB 2.3 ± 0.7 mm, residual bone 2.6 ± 1.2 mm).

Out of 52 implants evaluated with RFA, 19 were placed in BCP, 18 were placed in DPBB and 15 were placed in residual bone. The mean ISQ values were; BCP 68.5 ± 5.1, DPBB 70.4 ± 4.3 and residual bone 64.2 ± 7.1. There was a significant difference in mean ISQ values between implants placed in DPBB and residual bone (P = 0.008) but not between any other groups.

Paper IIIAfter 10 years of functional loading, the cumulative survival rate of the implants was 86.1%, based on 108 placed implants, 15 implant failures in 6 patients and additional 25 unaccounted for implants in 6 drop-out patients (Table 3). Nine of the failed implants were placed in grafted bone and 6 were placed in adjacent host bone.

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table 3. Life table of implants in paper III

time implantsenteringinterval

failedimplants

in interval

unaccounted for implants

csr (%)

Placement to loading 108 9 0 91.7

Loading to 1 year 99 1 0 90.7

1-2 year 98 5 0 86.1

2-3 year 93 0 20 86.1

3-5 year 73 0 1 86.1

5-10 year 72 0 3 + 1 sleep 86.1

At 10 year 68 86.1

CSR = cumulative survival rate

The mean pocket depths were 3.4 ± 1.5 mm, 3.6 ± 1.9, and 3.3 ± 1.4 mm, for all implants, implants placed in host bone and implants placed in grafted bone, respectively. None of the implants was found to be clinically mobile. The mean ISQ values (at implant level) for all implants were 69.7 ± 7.5 (range; 55 - 89), for implants placed in host bone were 68.3 ± 6.7 and implants placed in grafted bone were 70.2 ± 7.8. There were no significant differences in pocket depth or ISQ values between implants placed in host bone compared with implants placed in grafted bone (p=0.59 and p=0.34, respectively) and not between smokers or non-smokers (p= 0.19 and p=0.95, respectively) at 10-years follow-up.

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Paper VThe clinical data of subjects and surgical sites are presented in Table 4.

table 4. Clinical data of subjects and surgical sites. Ed, edentulous; max, maxilla; part, partly; mand, mandible.

subj sex age region healing time (months)

n of implants

Graft implant

1 M 57 Ed. max. 8.2 3.0 6

2 M 66 Ed. max. 8.1 3.3 6

3 F 58 Part. ed. mand. 8.2 2.9 4

4 M 66 Ed. max. 8.3 3.0 6

5 F 42 Part. ed. max. 8.2 2.8 6

6 F 66 Ed. Max. 8.2 3.0 6

7 F 68 Ed. mand. 8.1 3.0 4

8 M 67 Ed. mand. 7.9 5.7 4

9 M 67 Ed. max. 7.9 3.6 6

10 F 75 Ed. max. 8.1 3.0 5

11 M 59 Ed. max. 8.2 3.1 6

12 F 64 Ed. max. 7.9 3.8 6

13 F 29 Part. ed. max. 8.1 3.1 2

14 M 50 Part. ed. mand. 8.2 3.0 4

total 71

Mean (sd)

59.6 (12.1)

8.1(0.1)

3.3 (0.7)

range 29-75 7.9-8.3 2.8-5.7

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radiOGraphic resultsMarginal bone level/lossPaper II The mean marginal bone level after 3 years of loading was 1.32 ± 1.1 mm in the BCP group, 1.1 ± 0.78 in the DPBB group and 2.1 ± 1.3 for the implants placed in residual bone. There was a significant difference in marginal bone level between implants in the DPBB group and implants in residual bone and a significant difference in bone level within the groups between base-line and 3-years of loading.

Paper IIIThere were no significant differences in marginal bone levels between implants placed in host bone (1.8 ± 1.1) or grafted bone (1.5 ± 0.9) (p=0.16) and not between smokers (1.4 ± 0.7 mm) or non-smokers (1.6 ± 1.1 mm) (p=0.89). There was a significant marginal bone loss between 3 years and 10 years (p< .001), mainly in smokers who experienced a significant increase in marginal bone loss (p< .001). The marginal bone levels at different time points are presented in Figure 7.

figure 7. Marginal bone levels (mm) at the time of abutment connection (base-line) and after 1, 3, 5 and 10 years of functional loading.

The overall marginal bone level at 10-years follow-up was 1.8 ± 1.1.

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Graft dimension changesPaper IIIThere was a significant reduction in graft height (mean 2.5 mm, 16%) between 3 months and 2 years after grafting but no further reduction up to 10 years after grafting. There were no significant differences in length and width in any of the time periods.

Paper VThe volumetric reductions of the grafts after 7.5 months of healing were extensive and there was no significant difference in reduction between 90:10 (DPBB: AB) (55.3%) and 60:40 mixture (53.8%) (Figure 8). However, there was a significant difference in width reduction between the 90:10 and 60:40 mixtures when measured 3 mm from the top of crest, 46.9% (2.7 mm) and 37.0% (2.0 mm) respectively (P = 0.021) (Figure 9). The graft reduction in one patient is illustrated in Figure 10.

figure 8. Volume of the grafts at different time points, measured on CB/CT. CI, confidence interval; NS, no significant difference

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figure 9. Width of the alveolar crest at different time points, measured on CB/CT, 3 mm from the top of the alveolar crest. CI, confidence interval. NS, no significant

difference.

figure 10. 3-D reconstruction (CB/CT) of an edentulous mandible: A) preoperative, B) immediately after lateral augmentation, and C) after 7.5 months of graft healing.

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Health of the maxillary sinusesPaper IIFour (2 in each group) of the grafted sinuses presented with moderate mucosal swelling (22%) and 12 (78%) sinuses were judged to be healthy. No patients had any clinical symptoms.

Paper IIIIn 14 (64%) of the 22 grafted maxillary sinuses there were no signs of mucosal hypertrophy or fluid levels observed in the CB/CT scans. Five (22%) grafted sinuses presented with 1-6 mm swelling of the Schneiderian membrane and in 3 (14%) cases there were more than 6 mm of mucosal swelling.

histOlOGy and histOMOrphOMetric resultsPaper I (animal study)Seven out of 58 sites with dentin blocks presented moderate to intense inflammation. Inflammation was found in the groups with dentin blocks demineralized for 2 hours (N=2), 6 hours (N=2) and 12 hours (N=3), respectively. Since this was considered a surgical complication rather than a reaction to the dentin blocks, subgroups were created excluding sites with moderate to intense inflammation.

There was no significant difference in bone formation between the experimental groups. However, there was significantly more bone formation in both the 6- and 12-hour demineralization groups when the moderate and intense inflammation sites were excluded. Resorption increased with increasing time of demineralization without exclusion of the moderate and intense inflammation sites and was significantly higher after 6 hours of demineralization or longer (Figure 11). In the non-demineralized dentin group there were no signs of resorption or bone formation. There were no differences in inflammation or encapsulation between the demineralization groups.

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figure 11. Dentin onlay (D) deminera-lized for 6 hours. Bone formation (B) towards the calvarium (C) and surface resorption (arrows) of the deminerali-zed dentin. Less extensive resorption is seen towards the periosteum (P).

Paper IIIn general, the major impression was that the BCP specimens demonstrated a more pronounced and on-going inflammatory response (mostly macrophages and plasma cells) compared to the DPBB samples, even though inflammatory cells also were observed in relation to the DPBB particles, 3 years after sinus floor augmenta-tion. BCP particles, featured sharp edges and were at different levels of dissolution, most frequently observed at the edges of the particles, but occasionally the entire particle was partly dissolved (Figure 12).

figure 12. Light micrographs of BCP particles, 3 years after sinus floor augmenta-tion, under different levels of dissolution (black arrows) (A) with particles well inte-grated in bone (B). BCP, BoneCeramic; ST, soft tissue.

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In some cases the partly dissolved BCP particles were infiltrated by newly formed bone. In contrast, the DPBB particles, larger in size with smother edges, remained intact and showed no signs of resorption. DPBB particles were often in close apposition to bone of different quality, i.e. both immature as well as lamellar bone. However, the bone in close contact to BCP particles often lacked lamellae, the osteocytes presented with empty lacuna or pycnotic nuclei and sometimes the bone was osteoid like without osteoblast seams which may be interpreted as delayed or disturbed bone formation. There were no significant differences in any of the histomorphometric variables (Table 5).

table 5. Histomorphometric results after 3 years of graft healing.

bcp dpbb p-value

Particle area (%) ±SD 20.4 ±7.5 24.0 ±13.5 0.89

Bone area (%) ±SD 28.6 ±14.3 31.7 ±18.0 0.67

Soft tissue area (%) ±SD 51.0 ±15.0 44.3 ±10.5 0.35

Particle: bone contact (%) ±SD 37.8 ±10.9 44.4 ±12.1 0.47

Paper IVEleven years after sinus floor augmentation the DPBB particles were often well integrated and surrounded by lamellar bone, with no obvious signs of resorption (Figure 13), even though some multi-nucleated giant cells (MGC) occasionally were detected in close connection to particle surfaces facing the bone marrow. Macrophages and plasma cells were also observed. The histomorphometry of different tissues occupying the specimens are presented in Table 6.

table 6. Fraction of different tissues and particle: bone contact 11 years after sinus floor augmentation with 80% DPBB and 20% AB.

dpbb

DPBB area (%) ± SD 17.3 ±13.2

Bone area (%) ± SD 44.7 ±16.9

Soft tissue area (%) ± SD 38.0 ±16.9

DPBB: bone contact (%) ± SD 61.5 ±34.0

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The size of the particles varied considerably in all specimens (Figure 14). The results of the measurements of the particle lengths and areas are presented in Table 7. There were no statistically significant differences between the length and area of the particles after 11 years compared to those measured after 6 months (length p= 0.95, area p= 0.59) or the particles from the manufacturer (length p= 0.56, area p= 0.70).

table 7. The numbers and sizes of the histologically measured DPBB particles after different healing times.

time of biopsy number of particles

Mean length (mm) ± sd

Mean area (mm2) ± sd

0, from manufacturer

139 0.34 ± 0.18median 0.21

0.065 ± 0.060median 0.045

6 months 163 0.30 ± 0.24median 0.31

0.061 ± 0.055median 0.053

11 years 118 (length)/ 150 (area)

0.34 ± 0.22median 0.30

0.063 ± 0.051median 0.047

Paper VWhile DPBB particles were easily recognized and frequently present, the AB particles were sparse, mostly resorbed and difficult to recognize. All biopsies demonstrated bone and soft tissue integration of DPBB particles and inflammatory cells were frequently observed, irrespective of mixture of DPBB:AB, i.e. 90:10 or 60:40. The surfaces of the DPBB particles facing soft tissue were commonly lighter stained and in close relation to these areas MGCs were often observed. There were no obvious signs of DPBB particle resorption.

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figure 13. Light micrographs of ground sections of specimens taken 11 years after sinus floor augmentation with 80:20 DPBB:AB. DPBB particles are well integrated in dense bone (A) and in trabecular bone (B) without apparent signs of resorption.

figure 14. Histologic sections showing the similarities in DPBB particle size (and large particle size variations) after different healing times A) 6 months, B) 11 years and C) pristine particles from the manufacturer. *= DPBB particles. A and B are from the same patient.

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There were no significant differences between the 90:10 mixture and the 60:40 mixture in the fraction of different tissues (i.e. bone, DPBB and soft tissue) (Figure 15) or in any of the histomorphome-tric variables analysed.

figure 15. Fraction of different tissues 8 months after lateral ridge augmentation. NS, no significant difference; CI, confidence interval.

In both mixtures, there was no significant difference in fraction of DPBB between the graft facing residual bone and the graft facing periosteum; however, there was a significantly higher fraction of soft tissue and a lower fraction of bone formation in the grafts facing the periosteum (Figure 16). The histomorphometrical results also revealed that DPBB particles had a significantly higher degree of integration in newly formed bone closer to the residual bone compared to particles closer to the periosteum regardless of DPBB:AB mixture.

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figure 16. Results from the histomorphometrical analyses 8 months after lateral ridge augmentation comparing mean percentage of bone area, soft tissue area and DPBB area of the graft facing residual bone (bone side) and periosteum (periost side), re-spectively, of the two different compositions; 60% DPBB and 40% AB (60:40), and 90% DPBB and 10% AB (90:10). There was no significant difference in percentage of DPBB between the graft facing residual bone and the graft facing the periosteum in neither of the compositions, but there was a significantly higher fraction of bone formation and a lower fraction of soft tissue in the grafts facing the residual bone.

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discussiOn

The purpose of this thesis was to study the short- and long-term tissue reactions, graft resorption and clinical outcome after grafting with bone substitutes. This was performed using 3 different bone substitutes: i) dentin; ii) BCP; and iii) DPBB, at times combined with AB of different percentages. The incorporation of dentin block onlays with different degrees of demineralization and the tissue reaction was evaluated in the rat skull (study I). The graft incorporation of and tissue reaction to BCP and DPBB and the success rate of dental implants with moderately rough surfaces in the grafted area were evaluated 3 years after sinus floor augmentation in humans (study II). The clinical outcomes of dental implants with machined surfaces were analysed after 10 years of functional loading following sinus floor augmentation with DPBB in humans (study III). In addition, the long-term tissue reactions to DPBB and determination of the resorption rate of the grafted particles were evaluated after 11 years (study IV). In more challenging clinical situations, the graft incor-poration, tissue reactions and graft reduction with different DPBB and AB compositions were evaluated 8 months after lateral ridge augmentation in large edentulous defects in humans (study V).

paper iComments on material and methodsDentin has been used in several experimental studies as bone substitute due to its osteoconductive and osteinductive properties.15,

107, 182, 183 In a clinical situation the optimal graft would allow rapid bone incorporation to stabilize the graft and slow replacement by bone in order to maintain graft volume. Thus the speed of resorption

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of the bone substitute must be balanced with the ability of the patient to form new bone. Non-demineralized dentin is less prone to bone formation and the resorption starts later compared to totally demi-neralized dentin when placed in muscle pockets in rats.107 On the other hand, totally demineralized dentin may be resorbed too fast resulting in insufficient bone volume.

The present study was undertaken to evaluate the influence of partial demineralization of xenogenous dentin blocks on bone formation. The dentin blocks were demineralized in neutral buffered 24% EDTA between 1 and 12 hours before grafting to the rat scull. EDTA at neutral pH was chosen since it selectively removes mineral from the dentin surface without interfering with the integrity of the collagen matrix, which is important to preserve bone formation capacity, unlike the effect of etchants operating at lower pH.184 After preparation, the dentin blocks were stored in -18°C for one to 2 months. It has been demonstrated that dentin implants stored for more than 8-12 weeks seemed to lose some of their bone-inducing capacity, especially if stored in room temperature.185 Furthermore, radiation, heat and ultrasonic vibration reduce the capacity of dentin to induce bone formation.185 Hence, there was no further intention to sterilize the dentin implants in the present study, even though sterilization in ethylene oxide may have been a better alternative since this process does not seem to reduce inductive properties of demineralized bone.186 In the present study the dentin blocks were placed in a subperiostal pocket on the rat scull. If dentin particles are mobile during the osteogenic phase of bone healing, fibrous tissue is frequently formed, compromising proper graft incorporation due to fibrous encapsulation.187, 188 In contrast, when a bone block graft is placed in a tight pocket after subperiosteal dissection it seems stable and incorporates with the recipient bone.189

The periosteum plays an important role in bone formation and when lifted up from the underlying bone, the physical tension produced on the periosteum results in new bone formation.190 This is the reason why subperiosteal elevation was chosen as one of the controls besides non-demineralized dentin blocks. Abrahamsson et al191 evaluated a new technique to expand soft tissue using a subpe-riostally placed self-inflatable expander. They found new bone formation on the edges of the expander where the periosteum had

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been slowly raised. In the present study the same bone formation was found at the edges of the dentin blocks, interpreted as a result of osteoconduction due to the empty space created by the tenting of the periosteum. Holm-Bonferroni multiplicity adjustment, used in the present study, reduces the overall risk of falsely rejecting the null hypothesis (family-wise type I error). However, at the same time it increases the risk of type II errors, i.e. failing to reject a false null hypothesis. This was balanced by choosing an overall significance level of 0.1 instead of the traditional significance level of 0.05.

Comments on resultsIn the present study, the bone formation increased significantly with increasing degree of demineralization, provided inflammation was compensated for. The few sites (7 out of 58) presenting with moderate or intense inflammation were excluded in a subgroup since they were considered dependent on a surgical complication and/or lack of proper sterilization in order to isolate the effect of partial demineralization. Antibiotic treatment and proper steriliza-tion of the dentin blocks may have decreased the number of inflam-mation sites. The immunogenic properties of the tooth are related to the periodontal ligament and pulp182 which had been removed, but it may be speculated that the demineralization exposed more of the matrix which may have induced an immunologic reaction leading to a higher inflammation rate as this reaction was not present in the non-demineralized control group where the dentin implants had not been sterilized. This is in accordance with the results by Andersson and co-workers,182 who grafted non-demineralized dentin blocks to created defects in the mandible and the tibia of rabbits. Despite the only cleaning process was to place the dentin blocks in 1% chlorhex-idin for 10 minutes, they found no signs of inflammatory infiltrates in grafted sites after a healing period of 3 months. Another reason for inflammation in the demineralized dentin sites may have been remaining parts of EDTA indicating the need of a more elaborated cleaning procedure. However, the majority of the demineralized dentin sites did not present any moderate or intense inflammation.

Increase in bone formation (in sites with no or negligible inflam-mation) and dentin resorption (in all sites including moderate and intense inflammation) was seen with demineralization of 6 hours

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or more, although not coupled in the same statistical data set. This indicates that absence of moderate and intense inflammation may have a positive effect on bone formation192 and presence of inflam-mation most likely contributes to increased resorption.193 In the control group of the present study, where subperiosteal dissection alone was performed, bone formation was negligible. This may be due to the absence of a space holder. In further studies with similar designs the need of subperiosteal dissection only as a control seems to be superfluous, making the statistics less comprehensive and decreasing the sample size needed.

The results of the present study suggest that partial demine-ralization may provide a method for optimizing integration of dentin onlays which may serve to stabilize the graft, slowing down replacement resorption, thus preserving the volume of the graft.

paper iiComments on material and methodsThe purpose of this 3-year prospective study was to evaluate the tissue response to BCP after 3 years of healing and to evaluate the implants placed in the grafting materials after sinus floor augmenta-tion using DPBB as control. DPBB was used as control because of its extensive clinical and histological documentation.63, 71, 74, 194 Eleven patients were included in the study. A power calculation revealed that 10 patients were satisfactory for detecting a difference of 15% for paired morphometrical analyses with α=0.05 and a power of 80%. After 3 years, 2 patients did not want to participate in the biopsy retrieval. Out of the 18 biopsy cores retrieved, 10 specimens could be used since the rest contained few or no graft particles or were not available for paired tests. Due to the limited sample size and the limited number of dental implants placed (24 in sites grafted with BCP, 23 implants in sites grafted with DPBB and 15 in non-grafted sites) the histomorhometrical and clinical results must be interpreted with caution. After a healing period of 3 years, the buccal plate (where the augmentation had been performed) was almost intact and the presence of dental implants made the biopsy retrieval complicated. Computed tomography for three-dimensi-onal guidance might have facilitated the biopsy retrieval and would probably have resulted in more adequate histological samples from the grafted area.195

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Comments on resultsThe advantages of resorbing bone ceramics are multifactorial: i) foreign bodies are eliminated; ii) presence of the ceramic does not influence remodelling of the newly formed bone and finally; iii) the remodelled bone is stronger than the combination of ceramic and newly formed bone.99 In the present study, after 3 years of healing, the BCP particles were still at different levels of dissolution and demonstrated a more pronounced inflammatory reaction compared to DPBB particles. In addition, multinucleated giant cells often surrounded non-integrated BCP particles. The interface between CaP ceramics and the surrounding tissue has been described as dynamic, due to the fact that the biological apatite precipitation during dissolution of the material is continuously associated with cell colonization, adhesion, phagocytosis, osteoclastic resorption, bone ingrowth and bone remodelling.196 This description may explain the different cellular response between BCP and the less resorbable DPBB particles. Interestingly, the inflammation did not seem to affect the amount of bone formation or bone to particle contact, since there were no significant difference between BCP and DPBB regarding those parameters. The inflammation around the BCP particles may have been moderate enough not to have a major influence on bone formation. However, it could explain the presence of areas with delayed or disturbed bone formation close to the BCP particles. In the early healing phase bone formation seems to take place mainly between the BCP particles rather than on the surfaces themselves62, 101, 152 resulting in a lower bone to particle contact compared to DPBB.101, 157 This may be due to the fact that an increased percentage of TCP (40% in BCP) leads to reduced attachment of viable cells (i.e. osteoblasts and osteoclasts/monocytes) on the CaP surface shown in comparative in vitro studies of BCPs with different HA/TCP ratios.197, 198 Furthermore, it has been speculated that the reason for differences in bone to graft contact may be influenced by differences in structure and total surface area available and/or differences in bone formation characteristics between the investi-gated materials.157 However, in the current study, after a healing period of 3 years the same bone to particle contact was found in both BCP and DPBB, which may be explained by the degradation of TCP with time and a higher surface porosity which is more attractive for

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cell adhesion. Despite the dissolution of several BCP particles, there was no significant difference between the area percentage of BCP and DPBB. The equipment used in the present study does not allow histomorphometric measurements within the individual particles and the method used is probably too blunt to detect and measure partly dissoluted particles.

Even though there was an apparent difference in tissue reactions according to the histological findings, there were no significant differences in clinical outcome of the dental implants and the peri-implant tissue, i.e. PI, BoP, SBI, pocket depth or marginal bone level between the BCP group and the DPBB group. This raises the question of whether the on-going inflammatory response due to graft resorption has any long-term clinical relevance. However, well designed studies with a larger number of patients and long-term follow up are necessary to confirm these results.

papers iii and iVComments on material and methodsThe purposes of these studies were to evaluate the long-term (10 years) clinical outcome of dental implants placed in the posterior maxilla after sinus floor augmentation with DPBB (study III) and to histologically evaluate the long-term tissue response to DPBB particles and to determine their possible resorption (study IV).

In a recent systematic review of survival of implants placed in combination with sinus floor augmentation Pjetursson et al74 stated that longitudinal studies with observation periods of 10 years or more are completely lacking. Even though the number of patients are limited in the present studies it is important to report long-term clinical and histological outcomes of oral implants placed in areas augmented with BioOss®, the most used and documented bone substitute in the implant market, to guide clinicians and patients in their decision of choosing grafting material. A major drawback when conducting long-term clinical studies are withdrawn or dropped-out patients. Survival analyses presented as CSR statistics are often based on the assumption that the followed patients and implants have the same clinical outcome as the dropouts, an assumption that renders itself to critical discussion. In study III 6 (30%) of 20 patients dropped-out after 10 years which certainly may have influenced the

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results. However, Herrmann et al199 demonstrated that a random withdrawal of up to 50% of the original data did not influence the statistical outcome but this may have depended on a large patient sample and that drop-outs were random and not selectively appearing. In the present studies there was no attempt to match the gender of the consecutively included patients resulting in 14 women and 6 men. It may be speculated that this may have had an impact on the results, however, the gender does not seem to be a significant predictor of implant failures after sinus floor augmentation.200, 201

Another disadvantage in long-term follow up studies is that due to development and exchange of technical devices like radio-graphic equipment, those may not be in use during the entire study period. In study III the graft dimensions were measured on Scanora tomograms (height and width) and panoramic films (height and length) at 3 months, 1 year and 2 years after graft healing whereas after 10 years of graft healing all measurements were performed on CB/CT scans. The height and width are reliable measures since they were performed on Scanora tomograms, but the length of the graft measured on panoramic films may be less reliable due to variations in the magnification. However, if the patient is correctly positioned in the unit and the form of the object is rounded, the distortion is less marked.202 Furthermore, the CB/CT scans were used as guidance during biopsy retrieval which was essential since the lateral sinus wall was intact (i.e. no signs of grafting material). In addition, care was taken not to interfere with the oral implants and biopsies that had previously been retrieved from the same area. Despite apparent grafting material present on CB/CT, one out of 11 specimens did not contain representative material adequate for histologic and histomorphometric analyses due to difficulties in recognition of the graft and potential problems with the biopsy technique used. A region of interest (ROI) was chosen within the grafting material, representative for the entire biopsy, excluding the cortical (buccal) layer in order to avoid incorrect histomorphometrical values.

Comments on resultsNine (30%) of the originally 30 grafted maxillary sinuses showed perforations of the Schneiderian membranes. This complication rate is in accordance with other studies on sinus floor augmentation.200, 203

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It has been suggested that such perforations may be protected by a resorbable membrane204. In the present studies, Tisseel® was added to the composite of DPBB and AB to make the graft easier to handle and to prevent the particles from penetrating the perforations and no further measures were performed to close the perforations. The effect of sinus membrane perforations on implants survival is unclear in the literature. Some authors found that perforation did not compromise the survival rate of implants,205, 206 whereas others found a lower survival rate for implants placed in sites with perforated sinus membranes.207

Fifteen out of 108 placed dental implants failed giving a survival rate of 86.1%. However, on a per-patient basis the implant failure rate was 30% (6 out of 20 patients). Two of these patients, both heavy smokers, lost 10 of totally 15 failed implants. The cluster effect indicates that heavy smokers should be restricted from sinus floor augmentation procedures. These suggestions are in accordance with a risk factor analysis following maxillary sinus augmenta-tion using mainly DPBB (75.5% of the cases) in a retrospective multicentre study.200 The authors investigated 106 patients treated with 144 sinus floor augmentations and 328 implants with a mean follow-up of 48.4 months. They found that heavy smokers (more than 15 cigarettes/day) had significantly reduced implant survival. Furthermore, all 23 implant failures occurred in the first 18 months. Similarly, in the present study, there was no implant failure after 2 years of functional loading. Moreover, early failures may be due to insufficient initial stability, jaw bone quality 3/4 and jaw bone shape D/E according to the Lekholm & Zarb208 index209 even though chemical and structural changes of implant surfaces may have improved success rates. One inclusion criteria in the present studies was that the residual bone height should be ≤ 5 mm. In such a situation the main bone height to achieve implant stability is supported by the sinus floor graft. After a mean healing period of 6.7 months, biopsies were retrieved from the grafted sinuses. Histomorphometry revealed that sections from the augmented area were occupied by 21.2% lamellar bone and 10.2% woven bone.16 However, after a healing period of 3 years the surface area of lamellar bone had increased to 50.7%,77 indicating that the bone quality is improved with increasing healing time. These indications

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are supported by Handschel and co-workers23 who performed a histomorphometric meta-analysis of sinus floor augmentation with various grafting materials. The authors demonstrated that the TBV (determined as the percentage of the section consisting of bone tissue) increased significantly during time. An increased healing time may have improved bone quality and subsequently, possibly contri-buting to a decreasing number of early implant failures in study III.

There were no changes in graft length and width from 3 months to 10 years after sinus floor grafting in study III. However, there was a significant decrease in height between 3 months and 2 years with no further height reduction between 2 years and 10 years. After 2 years, the grafts seem to be stabilized with no further size changes. These results are in accordance with other studies evaluating vertical changes of DPBB for sinus floor augmentation.210, 211 Moreover, even if not systematically evaluated the radiopacity of the grafts appeared to be unchanged between 2 years and 10 years in panoramic radiographs. This may be explained by a dense packing of the DPBB particles137 during ingrowth of new bone and resorption of grafted bone particles, blood clot and Tisseel®. In study IV the resorption of DPBB was histomorphometrically evaluated. With respect to relevant tissues (bone, soft tissue and grafting materials) some authors have assume that DPBB is resorbed based on a smaller fraction of DPBB, measured by histomorphometry, compared to the ratio grafted DPBB.79, 86, 90 In none of these studies histomorp-hometry was performed at base-line, i.e. at the time of grafting or after the grafting material had been mixed prior to sinus floor augmentation. We performed histomorphometry on a sample of a mixture of 90% DPBB and 10% particulated bone mixed with human blood and Tisseel® that had not been grafted to a patient (Figure 17). Interestingly, the fraction of DPBB was only 26% and the majority of the sample contained blood which indicates that it may be incorrect to assume that a decrease in area DPBB fraction measured by histomorphometry after a healing period compared to the initial portion of grafted DPBB is an evidence of resorption.

Instead of area fraction, the length and area of the particles grafted to the sinus floor were compared at 6 months and at 11 years in the same patients to determine the possible resorption of DPBB in study IV. In addition the size of pristine particles from the manufacturer

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was measured. As previously stated, the entire skeleton is replaced by new bone after 10 years due to physiologic bone remodelling. However, in study IV there were no significant differences in length or area of the DPBB particles at any of the time points, indicating that resorption, if at all present, is very minor at least between 6 months and 11 years of graft healing. Furthermore, the DPBB particles were often well integrated in lamellar bone (Figure 18A) and the mean degree of DPBB to bone contact was 61.5 ± 34%. In some studies particle surfaces facing soft tissue have demonstrated a bright zone indicating superficial demineralization.79, 92 This is in accordance with the findings in study IV. In addition, multinucleated giant cells (not characterized as osteoclasts due to the absence of TRAP staining) were occasionally occupying these surfaces, however no signs of surface resorption, i.e. resorption lacunas, were evident in study IV (Figure 18B).

figure 17. Histologic sample of non-grafted 90:10 (DPBB:AB) mixture. Histomorpho-metry revealed 26% area fraction of DPBB.

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figure 18. Light micrographs of undecalcified ground sections of a biopsy taken 11 years after maxillary sinus floor augmentation with 80% DPBB and 20% AB. DPBB particles are well integrated in bone (A) and some particle surfaces facing soft tissue (ST) revealed a light zone (white arrows) in close contact to MGCs (black arrows) (B).

paper VComments on material and methodsThe purpose of study V was to radiologically and histomorphome-trically evaluate the graft reduction/resorption and graft incorpo-ration after lateral augmentation with 2 different compositions of DPBB and AB.

Since lateral ridge augmentation may be quite technique- and operator-sensitive, 212 it was decided to use one single surgeon for all clinical procedures. The number of patients in the present study is limited. However, a post hoc sample size calculation based on an alpha significance level of 0.05 to achieve 80% power to detect a clinically meaningful difference of 1.5 mm (SD 1.5) and 1 mm (SD 1.5) in width reduction between the 2 groups (90:10 and 60:40; DPBB:AB), suggested that 10 patients were enough to detect a difference of 1.5 mm (SD 1.5) and 18 subjects were sufficient to detect a difference of 1 mm (SD1.5), which is why we regarded 14 subjects to be an adequate number for the study. The fact that 10 jaws (8 maxillae and 2 mandibles) were edentulous and 4 jaws (2 maxillae and 2 mandibles) were partially edentulous may have affected the overall result, however the split mouth design compensated for this bias when comparing the 2 compositions.

DPBB is frequently mixed with AB in different ratios, varying from 0% to 50%, when used in GBR for lateral augmentation,

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to add osteoinductive properties.79, 80, 213-216 The optimal ratio of DPBB and AB regarding graft healing and reduction/resorption is not known. In the present study 10% AB was added to one of the compositions and 40% AB was added to the other composition. The 2 ratios were chosen from a clinical point of view since 10% AB can easily be collected with a bone scraper from the same neighbouring surgical site while 40% AB would require another surgical donor site when used for larger defects. In a recent clinical case series for lateral augmentation using DPBB and collagen membrane the time between grafting and re-entry was 9 to 10 months based on the authors experience and previous clinical and histological studies.217 In study V the healing time of 8 months was based on the same facts, however, a too long healing time may have interfered with the evaluation of the possible contribution of AB to the early graft incorporation. The width changes were measured on CB/CT scans not to be dependent on the surgical technique raising the flap and the integration of the graft. In addition, CB/CT scans allow for future long-term follow-up of volume- and width changes with time, without any further surgical intervention. In the current study the AB particles were sparse and mostly resorbed and difficult to distinguish from newly formed bone especially in lower magnifica-tion. When histomorphometry is performed from bone substitute grafted areas, commonly all bone (no distinctions are made between newly formed bone and the grafted bone particles) is included in the total bone area fractions presented in the literature.16, 77, 141, 142, 161, 218 For these reasons bone fraction presented in study V includes both newly formed bone and a small portion of residual AB particles.

Comments on resultsThere were no significant differences between the two graft compo-sitions regarding volumetric changes, ingrowth of newly formed bone and fractions of different tissues in the grafts (i.e. percentage of DPBB, bone and soft tissue). However there was significantly less width reduction 3 mm from the top of the alveolar crest, in favour of 60:40 (DPBB:AB) ratio compared to 90:10 ratio, The most important clinical outcome of the study was the possibility to install 71 of the planned 72 oral implants 8 months after lateral ridge augmentation with a mixture of DPBB and AB in large edentulous defects. Hence,

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the significant difference in width reduction of 0.7 mm between the two compositions seemed to be of less clinical importance.

The reduction of the grafts during the healing period in the present study was extensive, both regarding volume; 53.8% and 55.3%, and width; 37.0% and 46.8% (3 mm from the top of the crest) in 60:40 and 90:10 DPBB:AB compositions, respectively. These results are in contrast with the results from studies evaluating dimensional changes after sinus floor augmentation with DPBB, not exceeding 25% reduction of the graft.63, 70, 219, 220 There is a paucity of information concerning horizontal bone resorption of onlay grafts in the literature43. It is impossible to compare the results of graft resorption in the present study with most clinical studies due to lack of properly standardized radiographs and clinical measu-rements obtained immediately after grafting and after a healing period prior to implants installation. Hämmerle and co-workers217 reported a gain in width of 3.6 mm (± 1.5) in 12 patients after lateral augmentation with 100% blocks or particles of DPBB in localized defects after a healing period of 9 to 10 months. These results are in accordance with the results from the 60:40 mixture (3.5 mm) in the current study. Urban et al80 presented results from horizontal ridge augmentation in a case series of 22 patients. In 18 of the patients, particulated AB was mixed with DPBB in an unknown ratio. After a healing period of 8 months these patients had a width gain of 5.2 mm in the augmented area. The results in these 2 studies, however, lack information of the measurements immediately after graft augmentation making a comparison of graft reduction impossible. DPBB with or without collagen membranes have also been used to cover autogenous bone blocks to reduce graft resorption, decreasing the resorption rate to less than 10%,19, 221, 222 however, the frequency of complications may be increased.221

There were no signs of degradation of the DPBB particles in study V and the result from study IV indicated that DPBB may be without tendencies to resorption. It may be speculated, based on these indications in combination with the histomorphometric results from the non-grafted 90:10 mixture (Figure 17) that the reduction of the graft widths in study V is attributed to resorption of the AB, blood cloth and fibrin glue and soft tissue/denture pressure rather than resorption of the DPBB particles per se. In a radiographic study in minipigs it

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was recently demonstrated that the graft volume is better preserved with an increasing ratio of DPBB:AB after sinus floor augmentation. In study V there was no significant difference in volume between the 2 mixtures, despite a significant difference in width after 8 months of healing. These findings indicate that the graft was rather displaced after lateral augmentation and the two surgical sites (i.e. sinus floor versus alveolar ridge) have different prerequisites for analysing volumetric graft behaviour and biological healing of different grafting materials. It has been suggested that DPBB decelerate early bone incorporation134-136 leading to less resistance to soft tissue pressure in the early healing phase, which may explain the difference in width changes between the 2 compositions in favour of 60:40.

The histomorphometrical analyses revealed significantly less bone formation closer to the periosteum compared to the graft portion closer to the residual bone. Clinically this was experienced when the flap was raised at re-entry occasionally resulting in some grafting material attached to the soft tissue. This finding is in accordance with the results from a clinical and histological study evaluating the graft healing after DPBB had been augmented to fresh human extraction sockets.223 Histomorphometric analyses of biopsies retrieved after 9 months of graft healing showed a consistent increase of newly formed bone with a concomitant decrease in soft tissue area fraction from the most superficial histological sections to the deeper sections. The authors assumed that the superficial instability of the parti-culated graft probably produced the relatively high percentage of soft tissue area fraction. An extended healing time may have increased the bone ingrowth closer to the periosteum.174 The surgical procedure and choice of grafting material presented in study V is promising, however, further clinical and histological human studies addressing graft healing/reduction and implant integration, both short- and long-term, are needed to make more precise conclusions of the optimal ratio of DPBB and AB used for lateral augmentation of the alveolar crest.

clinical applicatiOns and future perspectiVesIt has been proposed by Chiapasco and co-workers43 that priority should be given to those bone augmentation procedures which involve less risk of complications, are less invasive and simpler to

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perform and reach their goals within the shortest time frame. Bone substitutes may be a solution to minimize the risk of complication and to simplify the augmentation procedure due to the absence of donor sites. In the present thesis it has been demonstrated that dental implants placed in sites after sinus floor augmentation with DPBB and BCP present good long-term clinical outcome. It may be suggested that these 2 bone substitutes are good alternatives to AB in sinus floor augmentation and implant treatment. The use of DPBB in these indications has previously been proposed in the literature. However, there is a need for long-term studies with larger number of patients to fully support the use of BCP for sinus floor augmentation. Lateral ridge augmentation presents a more challenging biologic situation than augmentation to the sinus floor due to exceeding the skeletal envelope and outer factors like soft tissues and prostheses acting as negative forces on the graft. It may be speculated that the non-resorbable DPBB which furthermore seems to give less tissue inflammation than BCP may be favourable in lateral ridge augmen-tation. In the present thesis the use of DPBB mixed with AB for lateral ridge augmentation was successful for implant placement if the grafts were overbuilt, calculating with a reduction of approxima-tely 30-40% of the graft. However, mixing the material with blood, Tisseel® and AB may have a negative effect due to higher resorption rate and less resistance to pressure compared to DPBB alone; indicating that the addition of these materials may be minimized and instead the resorbable membrane necessary to cover and stabilize the graft should be attached using pins or screws. On the other hand, if less fraction of AB in the graft is used there is a need for longer graft healing times. In those cases a graft healing time of approxima-tely 8-10 months may be appropriate prior to implant placement. Well-designed clinical studies need to be conducted to support these hypotheses. Dentin seems to be an interesting alternative to other bone substitutes due to its osteoinductive properties. Partial demine-ralization may be an interesting method to support early graft incor-poration and slow down replacement resorption and consequently preserve the volume of the graft. However, there is a need to further investigate the optimal demineralization time and process and how to effectively sterilize dentin without negatively affecting the osteo-inductive properties before clinical trials can be conducted.

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cOnclusiOns

1. Partial demineralization of xenogenous dentin blocks may be a method for optimizing bone formation and the integration of dentin onlays in an osteoconductive environment, provided inflammation is controlled for.

2. A similar degree of bone formation and bone-to-graft contact for BCP and DPBB was found 3 years after maxillary sinus augmentation. The BCP samples demonstrated a higher degree of inflammation compared to the DPBB samples possibly due to dissolution. There was no significant difference in implant success rate for implants placed in maxillary sinuses augmented with BCP compared to implants placed in DPBB following 3-years of functional loading.

3. The first 2 years after placement of implants with turned surfaces in sites after sinus floor augmentation with DPBB and autogenous bone seem to be critical for implant survival. At 10-years follow-up, the remaining implants presented good clinical and radiological results regardless of smoking habits or implant sites (augmented or residual bone). There seem to be no changes in graft sizes between 2 years and 10 years after grafting.

4. DPBB particles were found to be well integrated in lamellar bone, after sinus floor augmentation in humans, showing no significant changes in particle size after 11 years. If at all present, resorption is very minor at least between 6 months and 11 years after grafting.

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5. Even though there were no histomorphometrical differences between the DPBB:AB compositions, there was significantly less width reduction 3 mm from the top of the crest with a mixture of 60:40 compared to 90:10. The DPBB particles had a significantly higher degree of integration in newly formed bone closer to the residual bone compared to particles closer to the periosteum in both DPBB:AB compositions.

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acKnOwledGeMents

I would like to express my deepest gratitude to all who have made this work possible, with special thanks to:

Tomas Albrektsson, my supervisor and friend, for sharing your fantastic knowledge and experience in the field of research, always with a positive attitude, for being a master in solving complicated linguistic problems and a very entertaining travel companion and for so generously having opened up your home to me during my studies and research in Gothenburg. After the completion of this work I might even consider becoming a GAIS-fan.

Mats Hallman, my daily supervisor and friend, for always being available, positive, dedicated and helpful during this thesis. You have generously supported me with research advice and explana-tions of data from your previous studies. As a colleague and as the present head of the department of Oral & Maxillofacial surgery you have encouraged me during all the studies and you have made it possible to initiate and finalize this work.

Carina Johansson, co-supervisor and master in interpretation of what can be identified by means of a microscope, for advice, comments and helpful assistance in the field of histology and histomorphometry.

Jonas Becktor, co-supervisor for your kindness, encouragement, practical support and proofreading.

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Per Vult von Steyern, Head of the department of Materials science & Technology, for your support and all practical arrangements in the field of bureaucracy.

Sven Lindskog, co-author, for your skilful theoretical and practical supervision in paper I.

Christer Lindgren, co-author, for his major contribution in paper II and interesting discussions.

Thanks to all colleagues and staff at “Specialisttandvården” at Gävle County hospital, especially:

Tomas Strandkvist, my friend and colleague for your encourage-ment, kindness, patience and endless support during this thesis, in daily work and on the golf course.

Hartmut Feldmann, friend and colleague for the inspiring discussions, your support and accepting a higher workload during my absence from the department.

Eva Larsson and Karin Edkvist Jonsson, two of my nurses, for your engagement in my research and without whom, all the logistics concerning the patients in this thesis would have failed. Thanks for your enormous patience.

Nobuko Sakai Inglund, for your skilful help with the radiographic measurements in paper V

Solveig Sundéen Pikner, for all the measurements of marginal bone level in paper III.

The staff, research colleagues and PhD students, with Marieann Högman as head, at the Centre for Research and Development, Gävleborg County Council with special thanks to:

Statisticians Hans Högberg, Karl-Erik Westergren and Per Liv for all statistical analyses and fruitful discussions.

Lennart Fredriksson for your skilful editorial help, stimulating discussions and proofreading, and Inga-Lill Stenlund for always being service minded and helpful with a big smile on your face.

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The staff at the department of Biomaterials in Gothenburg with special thanks to:

Petra Johansson and Maria Hoffman for excellent laboratory work and friendly assistance in the world of microscopes, histomorpho-metry and histologic imaging.

Lars Zetterquist, my friend and former co-worker. Many thanks for your inspiration in the beginning of my carrier of becoming a specialist in Oral & Maxillofacial surgery and for your initial parti-cipation in study III and IV.

Lars Andersson for introducing me to the field of science during my residency in Oral & Maxillofacial surgery in Västerås.

Abram Mordenfeld, my dear brother and personal IT-assistant. You are always there when I need you, both in personal and professional matters, constantly connected to internet.

Maud Hultin, my ex-wife and dear friend for your support, professi-onal advice and being an excellent mother to our beautiful children. All participants in these studies.

Last but not least, Isabelle and Josefine, my wonderful daughters and joy of my life. Thanks for your endless love.

This work has been made possible by generous support and grants from:

Folktandvården in Gävleborg AB The Centre for Research and Development, Uppsala University and Gävleborg County Council, SwedenGeistlich Pharma AG, WolhusenAstra Tech AB, Mölndal

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a arne MorD enfelD on tissue reactions to anD mue… · investigation using the Bone Harvest...

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