On healing of titanium implants in biphasic calcium

phosphate

by

Christer Lindgren

Linköping University Medical Dissertation

No. 1274

2

To my mother

for her endless support

3

Table of contents

ABSTRACT 5

ABBREVIATIONS 7

ORIGINAL PAPERS 9

INTRODUCTION 10

Bone 11

Grafting materials 13

Bone histomorphometry 16

Dental implants 16

Implant follow up 19

Back scattered electron imaging and energy dispersive

spectroscopy 19

Bone Tissue Engineering, growth factors and scaffolds 20

AIMS 22

MATERIALS AND METHODS 23

Patients and study outlines 23

Presurgical examination, inclusion and exclusion criteria 24

Implants 25

Grafting materials 26

Surgery 26

Follow-up 28

Histology 30

Electron microscopy and elemental analysis 31

Surface modification of biphasic calcium phosphate particles

and deproteinized bovine bone particles 31

Statistics 32

4

RESULTS 33

Histological, elemental, clinical and radiographic outcome

(Paper I-IV) 33

Histological results 33

Results of elemental analysis 37

Clinical results 37

Radiographic results 38

SEM-and PCR analysis (Paper V) 39

DISCUSSION 42

Comment on Aim, Materials and Methods and Results Paper I-V 42

CONCLUSIONS 48

ACKNOWLEDGEMENTS 49

REFERENCES 52

PAPER I

PAPER II

PAPER III

PAPER IV

PAPER V

5

ABSTRACT

Background Previously, autogenous bone was considered the gold standard for grafting procedures in implant

surgery. Today, the use of bone graft substitutes is an alternative for sinus lift procedures. Nevertheless, only a

few bone substitutes are well documented and can be recommended as an alternative to autogenous bone grafts.

Both deproteinized bovine bone (DBB) and tricalcium phosphate (TCP) are materials that are frequently used

and well documented. During the last years a novel biphasic calcium phosphate (BCP) has been introduced to

the market. Until now only a few studies have been published.

Aims Studies I, II, III and IV. The overall aim of the present thesis was to evaluate a new biphasic calcium

phosphate for bone augmentation of the maxillary sinus. Deproteinized bovine bone was used as a control.

Study V. The aim of this in vitro study was to evaluate the response of human osteoblast-like cells (HOB) to

nano-crystalline-diamond-particle-modified (nDP-modified) and un-modified (control) deproteinized bovine

bone (DBB) and biphasic calcium phosphate (BCP) scaffolds.

Materials and Methods Studies I, II, III and IV. The studies were based on 11 patients (six women, five men)

with a mean age of 67 years (range; 50 to 79 years). All patients showed severe maxillary resorption with less

than 5mm of residual alveolar bone in the floor of the maxillary sinus, which excluded conventional implant

treatment. Twenty-two maxillary sinuses were augmented with BCP on one side and with DBB acting as a

control at the contralateral side. Simultaneously with the augmentation procedure 22 microimplants were placed

inside the augmented materials. After 8 months of graft healing the microimplants and a surrounding bone core

were retrieved for histomorphometrical analysis (Paper I) and for energy dispersive spectroscopy (Paper II).

After retrieval of the microimplants, 62 conventional implants were placed and left to heal for 8 weeks before

rehabilitation with fixed prosthetic constructions. The conventional implants were evaluated clinically at baseline,

after 1- and 3 years of loading (Papers III and IV). After 3 years of graft healing 18 biopsies were harvested from

9 patients for histomorphometrical analysis (Paper IV).

Computerized tomography (CT) of the maxillary sinuses was performed after 3 years of graft healing to allow

examination of the recipient sites.

Study V. Nano-crystalline-diamond particle-modification of DBB and BCP particles was carried out through

different steps of preparation including grinding and ultrasonic techniques. Scanning electron microscopy (SEM)

was carried out after 24 hours and 3 days. Real time-polymerase chain reaction (PCR) was carried out after 3

days, 1 week and 2 weeks of incubation. The following osteoblast differentiation markers were analyzed:

alkaline phosphatase (ALP), osteocalcin (OC), bone morphogenetic protein type 2 (BMP-2) and integrin alpha

10 (ITGA 10).

Results In paper I, the results revealed a similar degree of bone formation and bone-to-implant contact around

sandblasted and acid-etched microimplants placed in sinuses augmented with BCP or DBB. No obvious signs of

resorption of the BCP and DBB particles were noticed. There was a significantly higher amount of DBB

particles in contact with newly formed bone compared to BCP (p=.007).

In paper II, the median Ca/P ratios (atomic %), determined from >200 discreet sites within residual graft

particles and adjacent bone were analysed. The difference between the median values for BCP and DBB and for

BCP-augmented bone compared with DBB-augmented bone were statistical significant (p=.028 in each case).

The reduction in Ca/P ratio for BCP over the healing period is consistent with the dissolution of β-TCP and

reprecipitation on the surface of calcium-deficient hydroxyapatite.

In paper III, the results revealed that no implant placed in residual bone was lost, one implant placed in BCP

was lost after 3 months of functional loading due to infection, and one implant placed in DBB was lost only a

few weeks after insertion due to lack of initial instability. The overall implant survival rate was 96.8%. Success

rates for implants placed in BCP and DBB were 91.7% and 95.7% respectively. No significant differences in

marginal bone loss were found around implants placed in BCP, DBB or residual bone respectively.

In paper IV, it was shown that after 36 months (range; 36 to 37 months) of functional loading the overall implant

survival rate was 96.8%. Success rates for implants placed in BCP, DBB and residual bone were 91.7%, 95.7%

and 86.7% respectively. No significant difference was found between implants placed in BCP, DBB and residual

bone. The corresponding histological evaluation after 3 years of graft healing showed BCP particles under

different levels of dissolution. Dissolution was mostly observed on the edges of the BCP particles but in some

cases the entire particle was dissolving. In contrast, DBB particles showed no signs of resorption. The percentage

of graft particles in contact with newly formed bone was not significantly different after 3 years of healing for

BCP and DBB.

In paper V, cellular responses were evaluated in terms of attachment and differentiation. SEM after 24 hours and

3 days of incubation disclosed similar cell attachment and spreading for nDP-modified and non-modified DBB

and BCP particles. Real-time PCR revealed significant up-regulation of mRNA expression of ALP and OC and

by HOB grown on nDP-modified DBB and BCP-particles after 1 and 2 weeks compared to non-modified

particles. A significant down-regulation of BMP-2 on nDP-modified DBB and a significant up-regulation of

BMP-2 on DP-modified BCP was disclosed for HOB in relation to un-modified particles. Cell adhesion marker

6

ITGA 10 showed significant down-regulation in the mRNA level for both nDP-modified groups after 2 weeks of

incubation (mDP-BCP (p<.01) and nDP-DBB (p<.05) compared to the non-modified materials.

Conclusion

It is concluded that BCP can be used for maxillary floor augmentation and later placement of dental implants

producing equal results to those for DBB. Nevertheless, the initial HA/β-TCP ratio in BCP might not be

optimized for cell adhesion, which can affect the early healing phase. Furthermore, the results indicate that BCP

is not optimized for gene expression in its present form and that nDP-modified BCP enhances the osteoblast

phenotype suggesting that these scaffolds are appropriate cell carriers, superior to non-modified BCP particles.

Surface modification of bone substitutes is a new interesting field in bone tissue engineering (BTE).

Nevertheless, it´s still unclear if the modification will have any clinical impact.

7

ABBREVIATIONS AB Autogenous bone

ALP Alkaline phosphatase

BC BoneCeramic

BCP Biphasic calcium phosphate

BMP Bone morphogenetic protein

BMU Basic multicellular unit

BSE Back-scattered electron

BTE Bone tissue engineering

C2C12 Mouse myoblast cell line

CaCO3 Calcium carbonate

CT Computerized tomography

Ct Calcitonin

DBB Deproteinized bovine bone

DBM Demineralized bone matrix

EC Endothelial cell

EDS Energy dispersive spectroscopy

FGH Fibroblast growth factor

FHA Flouro-hydroxyapatite

GSH Grafted sinus height

H2SO4 Sulfuric acid

HA Hydroxyapatite Ca10(PO4)6(OH)2

HCL Hydroclorid acid

HF Hydrofluoric acid

HOB Human osteoblast-like cell

HSC Hematopoietic stem cell

IGF Insulin-like growth factor

IL Interleukin

ISQ Implant stability quotient

ITGA Integrin alpha

8

MSC Mesenchymal stem cell

MSFA Maxillary sinus floor augmentation

nDP Nano-crystalline-diamond-particle

OC Osteocalcin

OPG Osteoprotegerin

PCR Polymeras chain reaction

PDGF Platelet-derived growth factor

PES Peracetic acid ethanol sterilized

PTH Parathyroid hormone

RANK Receptor activator of NFκB

RANKL Receptor activator of NFκB ligand

RFA Resonance frequency analysis

ROI Region of interest

SEM Scanning electron microscopy

SLA Sandblasted, large-grit, acid etched

TCP Tricalcium phosphate Ca3(PO4)2

TGF Transforming growth factor

TNF Tumor necrosis factor

TPS Titanium plasma sprayed

VEGF Vascular epithelium growth factor

9

ORIGINAL PAPERS

This dissertation is based on the following papers, which will be referred to throughout by

their Roman numerals (papers reprinted by kind permission of journal editors).

I Lindgren C, Sennerby L, Mordenfeldt A, Hallman M. Clinical histology of

microimplants placed in two different biomaterials. Int J Oral Maxillofac

Implants 2009; 24:1093–1100.

II Lindgren C, Hallman M, Sennerby L, Sammons R. Back scattered electron

imaging and elemental analysis of retrieved bone tissue following sinus

augmentation with deproteinized bovine bone or biphasic calcium phosphate.

Clin Oral Implants Res. 2010 Sep;21(9):924-30.

III Lindgren C, Mordenfeldt A, Hallman M. A prospective 1-year clinical and

radiographic study of implants placed after maxillary sinus floor augmentation

with synthetic biphasic calcium phosphate or deproteinized bovine bone. Clin

Implant Dent Relat Res. 2010 May 11. [Epub ahead of print]

IV Lindgren C, Mordenfeldt A, Johansson C, Hallman M. A 3-year clinical

follow-up of implants placed in 2 different biomaterials used for sinus

augmentation. Int J Oral Maxillofac Implants [Submitted]

V Lindgren C, Ying X, Hallman M, Steinmueller D, Ghodbane S, Krüger

Mustafa K. Surface modified biphasic calcium phosphate particles enhance

expression of bone markers in osteoblast-like cells. [In manuscript]

10

INTRODUCTION

Wearing a removable dental prosthesis as a substitute for remaining teeth is today considered

a social and functional handicap (Allen & McMillan 2003). Reduction of alveolar ridge due to

edentulism results in difficulty in fitting dentures and placing dental implants. Therefore,

reconstruction or bone augmentation sometimes is necessary. In less severe cases, placement

of tilted implants can be an effective alternative to reconstruction (Aparicio et al. 2001, Del

Fabbro et al. 2010) and in situations where the residual crest lacks width but has sufficient

height, narrow implants may be used (Hallman 2001). For cases showing lenient atrophy in

the posterior maxilla a technique with elevated sinus membrane and simultaneous implant

placement has been evaluated (Lundgren et al. 2004). After elevation of the sinus membrane

the compartment is filled with blood and trapped with the replaced lateral window. The blood

clot acts as an osteoconductive scaffold for ingrowth of bone building cells. This technique

has also been evaluated in more severe cases with successful results (Thor et al. 2007).

Another technique used for patients with moderate atrophy in the posterior maxilla is the

osteotome technique (Summers 1994). For cases showing severe atrophy in the posterior

maxilla, Cawood class V and VI (Cawood and Howell 1988), a number of different

techniques have been used and evaluated. For example, maxillary sinus floor augmentation

(MSFA) (Boyne and James 1980, Tatum 1986), interpositional grafts (Hallman et al. 2005,

Nyström et al 2009) and placement of zygoma-anchored implants (Higuchi 2000, Malevez et

al. 2004). In cases with three-dimensional lack of bone in the posterior maxilla the use of

veneer grafts prior to implant treatment has been evaluated (Bahat and Fontanessi 2001).

Ten to fifteen years ago, autogenous bone (AB) was the most used grafting material for

reconstruction of the floor of the maxillary sinus prior to implant surgery (Lundgren et al.

1996, Lundgren et al. 1997, Johansson et al. 1999, Wannfors et al. 2000). Autogenous bone

accelerates the healing process due to its osteoinductive properties by stimulation of bone-

inducing factors (Urist 1965). Furthermore, the risk of transfection is eliminated and there is

no cost for the material itself. As donor sites, the iliac crest, mandibular symphysis,

mandibular ramus and the calvarium of the skull have been used (Jensen et al. 1990, Hirsch

and Ericsson 1991, Lundgren et al. 1999, Tulasne 1999, Wannfors et al. 2000). Nevertheless,

there are some drawbacks using AB. The surgical procedure often demands treatment in

general anesthesia and a second surgical site is required with accompanying donor site

morbidity and risk of complications (Beirne 1986, Nkenke et al. 2001). Moreover, resorption

of autogenous bone grafts also represents a problem (Körlof et al. 1973, Johansson et al. 2001,

Sjöström et al. 2006). Hence, different bone substitute materials have been developed through

the years. The benefits of utilizing bone substitute materials are many. The surgical procedure

can be performed in local anesthesia, no donor site is required and the actual cost for the

surgical procedures decreases.

Bone substitute materials can be resorbable, non-resorbable or partially resorbable. The use of

a non-resorbable material will preserve the volume of the graft, which is an advantage

(Hallman et al. 2002a, Hallman et al 2002b, Hallman and Thor 2008, Hallman et al. 2009,

Mordenfeldt et al. 2010). Nevertheless, lack of osteoinductiveness leads to prolonged graft

healing times. Furthermore, for some xenogenic and allogeneic materials there is a possible

risk of transfection (Honig et al. 1999, Wenz et al. 2001). Hence, the use of a synthetic bone

substitute material is preferable to xenogenic and allogenic materials if the same results can

be obtained.

Bio-Oss® (Geistlich pharmaceutical, Wollhausen, Switzerland) consists of 100% bovine

deproteinized cancellous bone and is the most documented and used biomaterial in implant

dentistry. Bio-Oss®

particles have physical and chemical properties similar to human bone.

The porous structure allows ingrowth of blood vessels and bone building cells. However,

11

since it originates from bovine bone, the material has been questioned (Honig et al. 1999).

The most documented synthetic biomaterial is calcium sulfate that has a history of more than

100 years and still is used for sinuslift procedures (Pecora et al. 1998, de Leonardis and

Pecora 2000, Slater et al. 2008, Dashma et al 2009). The second most used synthetic

biomaterial is tricalcium phosphate, Ca3(PO4)2 (TCP) (Esposito et al. 2006). Recently a novel

biphasic calcium phosphate, BoneCeramic®

(BC) (Straumann, Basel, Switzerland) has been

introduced to the market, BC consist of 60% hydroxyapatite (HA), Ca10(PO4)6(OH)2 and 40%

β-TCP Ca3(PO4)2. In theory, the β-TCP part will dissolve into Ca and PO4 ions, which may

stimulate bone formation (Daculsi 1998), whilst the HA part probably not will resorb

(Hallman et al. 2001, Mordenfeld et al. 2010). The HA part will also act as a 3-dimensional

scaffold for ingrowth of bone building cells and blood vessels.

There is extensive ongoing research about dental implant surfaces. It has been shown in

experimental studies that surface modifications of the implant have an influence on bone

formation, but not much is yet known about the clinical impact (Wennerberg and Albrektsson

2009). However, research in implant dentistry focusing on the influence of biomaterial

surface modification on bone formation is sparse.

The interface between bone substitute particles and newly formed bone, importance of nano-,

micro- and macrostructure of the material and possible impact on bone reformation are a

novel chapter in bone biology. Furthermore, tissue engineering and surface treatment of

scaffolds is a new interesting field in science. Elemental analysis and analysis of gene

expression are new methods to be considered in order to more accurately evaluate the

interface of biomaterials and the progress of bone reformation.

Bone The human skeleton is the main supportive organ in the body, in which bone tissue constitutes

the major part. Bone is one of the hardest tissues in the human body and it protects vital

organs and contains bone marrow, where the blood cells are formed. Bone consists of mineral,

collagen, non-collagenous proteins, water and lipids. The interactions of these constituents

play major roles in determining the mechanical behaviour of bone (Cullinane & Einhorn

2002). Bone is both a living and adaptive connective tissue constantly undergoing changes

and represents a reservoir of Ca ions for the organism (Knothe ML et al. 2004). Bone includes

macroscopically a cortical (compact) and a trabecular (cancellous) component (Marks and

Odgren 2002). Cortical bone represents about 80% of the mature skeleton and consists mainly

of hydroxyapatite Ca10(PO4)6(OH)2. Trabecular bone consists of more soft tissue

(hematopoietic or fatty marrow) components and has a low content of minerals.

Microscopically, bone tissue is divided in lamellar (mature) and woven (immature) bone.

Woven bone has a more rapid turnover and is considerably weaker than lamellar bone since

the collagen fibres are not as tightly organized as in lamellar bone.

There are four different cell types found in bone. The osteoblast, the bone lining cell and the

osteocyte are derived from mesenchymal osteoprogenitor cells in bone marrow and

periosteum. The fourth cell type is the osteoclast, which is derived from multinuclear cells

(Mackie 2003).

The osteoblast

Bone is formed initially by osteoblasts synthesizing type I collagen fibres. The collagen fibres

represent 90% of the bone matrix, while the remaining 10% consists of collagen matrix

proteins (e.g. osteopontin, bone sialoprotein, osteonectin and bone acidic glycoprotein-75)

and non-collagenous matrix proteins (e.g. osteocalcin and bone sialoprotein). Moreover, the

osteoblasts also form amounts of growth factors, e.g. transforming growth factor-β (TGF- β)

and insulin-like growth factors (IGF-I, IGF-2) later used in the remodelling process (Karsenty

12

2003). Certain osteoblasts are located on the surface of the bone where they form a syncytium

and secrete the organic components of the bone matrix. Between the osteoblast and the

mineralized zone a thin layer of osteoid occurs (Lerner 2000). Some osteoblasts are contained

in the bone matrix and become osteocytes.

The bone lining cell

Bone lining cells are essentially inactive flattened cells mainly derived from osteoblasts. The

bone lining cells cover the non-remodelling bone surface and are connected with osteocytes

through cell processes into the bone. The cells can be activated and differentiated to

osteogenic cells and are suggested to function as a barrier for bone fluids and ions (Miller et al.

1989).

The osteocyte

Osteocytes originate from osteoblasts that have migrated into and become trapped and

surrounded by bone matrix that they produced themselves. The spaces they occupy are known

as lacunae. Osteocytes are the most numerous cells in mature bone and they communicate

with other osteocytes and with osteoblasts on the surface through gap junctions. Osteocytes

have also been suggested to act as mechano-sensory receptors detecting the mechanical

loading on the bone and through communication with osteoblasts increasing the bone volume

in the stressed area (Noble and Reeve 2000).

The osteoclast

The osteoclast is responsible for bone resorption and takes part in the calcium homeostasis of

the body. Osteoclasts are large multi-nucleated cells and originate from hematopoietic mono-

nucleated cells in bone marrow or from the spleen. The osteoclast belongs to the leukocyte

family and is equipped with phagocytic-like mechanisms similar to circulating macrophages.

They are located on bone surfaces called Howship’s lacunae or resorption pits. The

osteoclastic activity is controlled by different hormones in order to balance the serum calcium

level, e.g., parathyroid hormone (PTH), 1,25(OH)2-vitamin D3 and calcitonin (Ct). The

activation of osteoclasts is due to indirect regulation from the osteoblasts (Lerner 2000).

Bone remodelling Each year, ten percent of the skeleton is replaced in a healthy individual (Lerner 2006). The

bone resorption is performed by the osteoclasts and bone formation is carried out by the

osteoblasts in a complex process. In brief, the bone remodelling cycle involves resorption,

reversal and formation (Hill 1998, Hadjidakis and Androulakis 2006). The osteoblasts and

osteoclasts interact with each other in basic multicellular units (BMU), where the remodelling

phase takes place. It is estimated that the human skeleton has 1-2 x 106 such units (Riggs and

Parfitt 2005). It is not likely that actively bone-forming osteoblasts are the cells that activate

osteoclasts. Rather, inactive osteoblasts, either the lining cells or the pre-osteoblast, are

responsible, although this has not been definitively shown (Lerner 2006). In the BMU,

osteoclastogenesis is preceded by the expression of receptor activator of nuclear factor κB

ligand (RANKL), which in turn binds to the RANK receptors on the membrane of the

osteoclast-progenitor cell. This interaction promotes osteoclast activation (Wada et al. 2006).

Osteoblasts are known to produce osteoprotegerin (OPG), which can inhibit RANK activation

through blocking of RANKL on the osteoblast. Oestrogen and calcitonin are also inhibitors of

osteoclastic activity. Furthermore, the expression of RANKL is up-regulated by interleukin-1

(IL-1), tumour necrosis factor α (TNF-α) and vitamin D. The area about to be resorbed by the

osteoclasts is determined by PTH activation. Meanwhile, the osteoblasts degrade the thin

13

osteoid layer covering the bone through proteolytic enzymes (Lerner 2000). The osteoblasts

then withdraw from the bone surface, leaving the area for osteoclasts to begin the resorption.

In this area (sealing zone) the resorption is carried out by acidic degradation of mineral and

matrix. The osteoclasts are active during 3 weeks before moving to the next site. Growth

factors like IGF-I/II and TGF-β are released in the area stimulating osteoblasts for bone

building activity.

Grafting materials

Autogenous bone is frequently used for reconstruction or augmentation of the severely

resorbed posterior maxilla. As alternatives, there are numerous different bone substitutes on

the market with different properties and characteristics. Bone graft substitutes are divided in 3

groups: allogenic bone, xenogenic bone and alloplastic bone.

Autogenous bone grafts:

Autogenous bone (AB) originates from the same individual receiving the graft. AB is still

considered “gold standard” among the grafting materials since the graft is both osteoinductive

(contains both living cells and proteins which can stimulate new bone formation) and

osteoconductive (scaffold for bone building cells and blood vessels) in various degrees

depending if cancellous or cortical bone is used. Nevertheless, drawbacks have been reported

in terms of morbidity at the donor site, graft resorption and prolonged surgical time (Beirne et

al. 1986, Johansson et al. 2001, Nkenke et al. 2001).

AB grafts consist of bone marrow-derived osteoblastic cells and preosteoblastic precursor

cells that stimulate osteogenic properties, noncollagenous bone matrix proteins and growth

factors giving osteoinductive properties and bone mineral and collagen giving

osteoconductive properties (Khan et al. 2005).

Healing of autogenous bone grafts

The rate and extent of revascularization, graft orientation, the embryonic origin of the graft

and contents of local growth factors are determinants for a successful healing of a bone graft

at a recipient site (Alberius et al. 1996). The revascularisation process differs between cortical

and cancellous bone due to different morphologies. Cancellous grafts are revascularised more

rapidly and completely than cortical grafts. Cortical bone tends not to repair completely with

time; instead it exhibits an appearance of both viable and necrotic bone. Contrary to cortical

bone, cancellous bone tends to repair completely with time. Another difference between the

two types of bone is that cortical grafts undergo a creeping substitution process in comparison

to cancellous bone, which initially involves an appositional bone formation phase (Burchardt

1983).

Allogenic bone grafts:

Allogenic bone is harvested from another individual of the same species. There are different

forms of allografts available. Mineralized or demineralised bone, frozen or freeze-dried bone,

demineralised dentin and antigen-extracted allogenic bone have all been tested. Allografts are

osteoconductive but most likely have less osteoinductive capacity in relation to autogenous

grafts due to the absence of living cells (Becker et al. 1995). In implant dentistry the use of

frozen or freeze-dried allograft is sparse. However, more common is the use of commercial

allograft which has been treated with a demineralization or auto-lysis procedure. Commercial

allograft bone is both osteoconductive and is considered to have osteoinductive properties due

to maintained BMP (Bone Morphogenetic Proteins) activity (Urist 1965). In a recent in vitro

study (Bormann et al. 2010 fresh-frozen cancellous bone (native), peracetic acid-ethanol

sterilized (PES) cancellous bone, cortical bone and demineralised bone matrix (DBM) were

14

examined in respect of osteoinductivity. Cultivating C2C12 cells (mouse myoblast cells)

together with each bony allograft and later measurement of cell proliferation and alkaline

phosphatase activity revealed that proliferation was significantly enhanced by the native

cancellous bone. The osteogenic differentiation was significantly decreased for PES-sterilized

cancellous bone. Still, clinical studies comparing osteoinductivity of bony allografts are

lacking.

Healing of allogeneic bone grafts

In orthopedic surgery, allogenic bone grafts have been used for several decades. The

principles for incorporation of allografts follow the same principles as for autogenous bone

grafts but probably proceed more slowly due to the absence of living cells that are

osteoinductive, although allografts might have some osteoinductive capacity. Studies have

shown that human mineralized bone allografts could be successfully used for sinus lift

procedures before placement of implants (Gapski et al. 2006). The use of an ideal graft

material should result in the formation of a high percentage of vital bone after graft

maturation. The literature shows varying results for different grafting materials, with vital

bone content from 14 to 44%. In a study using mineralized cancellous bone allograft for sinus

augmentation a vital bone content of 25.2% was found after 9 months of graft healing (Froum

et al. 2005).

Xenogenic bone grafts:

Xenogenic bone originates from other species than humans. Examples are corals, bovine bone

and porcine bone. In order to obtain immunological safety, proteins have been extracted from

these materials using various procedures. Algipore®

(FRIOS) is a porous fluoro-

hydroxyapatite (FHA) manufactured from calcifying marine algae (Corallina officinalis).

Biomaterial processing involves pyrolytical segmentation of the native algae and hydrothermal

transformation of the calcium carbonate (CaCO3) into FHA (Ca5(PO4)3(OH)xF1-x). Studies

have shown that Algipore® is a suitable biomaterial for sinus grafting of severely atrophic

maxillae (Schopper et al. 2003).

The most used and documented bone substitute in implant dentistry is deproteinized bovine

bone (DBB) (Esposito et al. 2006). DBB consist of 100% deproteinized bovine

hydroxyapatite. The material has no osteoinductive properties but works as a 3-dimensional

scaffold for ingrowth of blood vessels and bone building cells (osteoconduction). In several

studies, the material has been shown to integrate well with bone (Hallman et al. 2001,

Schlegel et al. 2003, Kim et al. 2009). Despite in vivo studies showing favourable effects with

DBB, some animal studies have reported that DBB delays early bone formation (Araújo et al

2009) and does not enhance bone formation (Botticelli et al 2004). Nevertheless, in a review

article (Esposito et al. 2008) the authors concluded that DBB might be equally effective as

autogenous bone grafts for augmenting atrophic maxillary sinuses. Furthermore, DBB is

suggested to be used as replacement to autogenous bone grafting. However, these findings

need to be confirmed by large multicenter trials. DBB can either be used alone for various

augmentation procedures or in combination with autogenous bone. From a biological point of

view it could be an advantage to mix DBB and autogenous bone due to the osteoinductive

properties of autogenous bone. Nevertheless, the benefit has not been shown in clinical

studies. A review article (Jensen et al 2011) tested the hypothesis that there was no difference

when DBB or DBB mixed with autogenous bone was used as graft for maxillary sinus floor

augmentation (MSFA). The authors could not draw any conclusions concerning the matter

due to lacking of long-term clinical and radiographical studies.

The material has been questioned because it originates from bovine bone (Honig et al. 1999,

Wenz et al. 2001).

15

Healing of xenogenic bone grafts

In order to avoid immunological rejection after the grafting procedure most of the xenogenic

bone substitutes need to be deproteinized through various procedures. The osteoinductive

capacity disappears during these processes and the graft can therefore only act as an

osteoconductive scaffold. This leads to a slower formation of new bone compared to

autogenous bone grafts. However, collagenized xenografts of porcine origin have recently

been tested and evaluated in animal studies with promising results according to

biocompatibility, bioabsorbability and osteoconductivity (Calvo Guirado et al. 2011,

Fernández et al. 2011).

The use of xenogenic grafts like Bio-Oss® has been examined in various surgical sites such as

fresh extraction sockets and local defect areas of the alveolar crest, showing that appropriately

treated xenogenic bone is biocompatible and integrates well with recipient areas (Esposito et

al. 2006, Kim et al. 2009). Today, xenografts are frequently used for bone augmentation

procedures in implant dentistry due to their similarity to human bone.

Alloplastic grafts:

Alloplastic bone graft substitutes are synthesized alternatives to autogenous bone grafts, bone

allografts and xenogenic bone. Calcium-based ceramics, calcium-sulphate and bio-active

glasses are all examples of alloplastic bone graft materials with different compositions.

Furthermore, they also exhibit different biological and mechanical properties.

Alloplastic bone graft substitutes usually contain hydroxyapatite (HA) and different calcium

polymers such as, β-tricalcium phosphate or sintered calcium phosphates, bioglass or sintered

calcium sulphates. In contrast to HA, pure calcium phosphate or calcium sulphate is generally

weaker in its composition and will presumably dissolve chemically into calcium and

phosphate ions, which may stimulate bone formation (Daculsi 1998).

By dissolution of tricalcium phosphate (TCP) in naphthalene in order to create a uniform

crystal structure (100 to 300µm) with optimum pore size and further sintering, β-tricalcium

phosphates (β-TCP) are produced. In relation to pure calcium phosphate, β-TCP has a more

organized crystal structure but will still dissolve into calcium and phosphate ions. The

material can be used for maxillary sinus floor augmentation but drawbacks such as rapid

dissolution and volume reduction of the graft have been reported (Lu et al. 2002).

Cerasorb®

(Curasan AG, Kleinostheim, Germany) is the most used commercial β-TCP today

for dental implant surgery (Szabó et al. 2005).

In order to combine different properties, materials are sintered together. Biphasic calcium

phosphate materials contain a sintered mixture of HA and β-TCP. Bone Ceramic®,

(Straumann, Basel, Schweiz) aimed for the market of implant surgery consists of 60% HA

and 40% β-TCP, which give the material properties to obtain graft volume and possibly also

give the material bone stimulating capacity due to the dissolution of Ca and phosphate ions.

Short term histological follow-up has been published on biphasic calcium phosphate (BCP)

(Artzi et al. 2008, Cordaro et al. 2008, Lindgren et al. 2009, Friedmann et al. 2009). Still, long

term-follow up studies regarding histology and placed implant survival is still lacking.

Examples of other alloplastic materials are Easygraft CRYSTAL®

(Degradable Solutions AG,

Schlieren, Switzerland), Tricos®

(Baxter healthcare corp., Waukegan road, McGaw Parkil,

USA) and calcium sulphate (CaSO4) (Surgiplaster, Ghimas, Bologna, Italy).

Healing of alloplastic grafts

Whether placement of synthetic bone substitute material succeeds is dependent on the

environment in the recipient site. Formation of new bone can start if the material is placed in

close contact to bone, serving as a three-dimensional scaffold for ingrowth of blood vessels

and bone building cells.

16

Bone histomorphometry Histomorphometry is a quantitative histological examination of an undecalcified bone biopsy

performed to obtain information about bone remodelling and structure (Kulak and Dempster

2010). Histomorphometry has traditionally been assessed in two dimensions but in the last

two decades there have been advances in histomorphometric techniques with coupled

stereology software to facilitate the measurements (Malluche et al. 1982).

In clinical practice, bone biopsies are most often performed to exclude or confirm a diagnosis

but also in order to evaluate the healing process for different bone substitutes or implants. A

bone biopsy can either be harvested by needle (closed biopsy) or through the skin or mucosa

to expose an area of the bone (open biopsy). Earlier, a bone biopsy was harvested either by

hand or by using a trephine burr. Placement of microimplants is a new technique to construct

a miniature example of the titanium-bone interface (Jensen and Sennerby 1998, Lundgren et

al. 1999, Hallman et al. 2002b). In brief, simultaneously with sinus augmentation a titanium-

threaded microimplant is inserted in the residual bone penetrating the grafting material. After

a certain healing time the microimplant with a surrounding bone core is retrieved using a

trephine burr. The specimens are fixed by immersion in buffered formalin solution,

dehydrated in alcohol and embedded in plastic resin. A specialized laboratory prepares

undecalcified sections and histological and histomorphometrical analysis can be carried out

after the samples have been stained with different dyes.

Dental implants Osseointegration is described as the close contact between implant and bone (Albrektsson and

Wennerberg 2004). The interaction between bone and implant depends on various factors

such as quality and quantity of the bone, implant surface characteristics to facilitate cell

attachment and protein adsorption (Junker et al. 2009). Although today osseointegration is a

standard term in dental and medical literature, the interface between the implant surface and

the surrounding tissues is not yet fully explored (Engqvist et al. 2006).

Implant stability can be divided into primary and secondary stability (Sennerby and Roos

1998). Primary implant stability depends on implant design, density of the bone and surgical

technique. Secondary implant stability depends on primary stability, the used implant material

and tissue response.

A surgical trauma occurs when preparing an implant site in bone tissue. Erythrocytes, fibrin

clot and bone fragments will surround the inserted implant. In a histological study on rabbit

tibia, mesenchymal cells and multinuclear giant cells were present around the placed implant

after 7 days (Sennerby et al. 1993). Formation of woven bone occurred from the endosteal

surfaces towards the implant. Woven bone was also found in the collagen matrix in the

marrow compartment. Gradually the amount of bone increased and filled the implant threads.

The remodelling process was first seen after 6 weeks and was completed after 90 days.

As early as 1981, surface structure was identified as one factor important for implant

osseointegration (Albrektsson et al. 1981). Review studies have shown that an increase of the

surface roughness of the dental implants enhances osseointegration compared to dental

implants with smoother surface characteristics (Cooper 2000, Le Guéhennec et al. 2007).

Today, there are several techniques to alter surface topography of a machined surface. Some

techniques will remove particles from the surface and create a concave profile, such as:

electropolishing, mechanical polishing, blasting, etching and oxidation. Some techniques add

material to the metal, such as hydroxyapatite (HA) and calcium phosphate coatings, titanium

plasma sprayed (TPS) surfaces and ion deposition (Wennerberg and Albrektsson 2009). It is

unlikely that only one parameter can strengthen bone response around an implant surface. If a

surface is modified macroscopically the micro- and nano-roughness on the implant will also

change. For example, Straumann’s SLActive implant has not only a hydrophilic surface, but

17

its micro- and nano-roughness also differ from the SLA (sandblasted, large-grit, acid etched)

surface. Today, it is not known whether nano-roughness play an important role in

osseointegration as there are only a few in vivo studies available and they suffer from either

experimental study designs or poor control of the influence of other non-topographical surface

parameters (Wennerberg and Albrektsson 2009).

Blasted surfaces

In order to increase implant surface roughness different blasting procedures can be carried out.

Aluminium oxide and bioceramic particles are two commonly used blasting materials. In a 5-

year prospective study, two different surfaces on Astra Tech implants were evaluated

(Wennström et al. 2004). Astra Tech implants with either a TioBlast® (TiO2-blasted) surface

or a turned surface were evaluated in patients with periodontitis. No statistically significant

differences were found comparing the different implants regarding bone loss. These results

were in accordance with an earlier study (Karlsson et al. 1998) where Astra Tech TioBlast®

implants were compared with implants with a machined Astra Tech surface and evaluated

after 2 years of loading. No statistically significant differences were found regarding marginal

bone loss. A review article by Wennerberg and Albrektsson (2009) concluded that blasted

implants demonstrate better bone integration than turned/machined implants. In an animal

study by Duyck et al. (2007), a bone-stimulating effect was observed after 6 weeks for

implants with a rough blasted surface when compared with implants with a turned surface

placed in the tibias of six mature female white rabbits. In contrast to animal studies, clinical

studies often fail to find any major advantages or disadvantages with blasted implants when

compared with turned implants.

Etched surfaces

Hydrochloric acid (HCl), sulfuric acid (H2SO4) and hydrofluoric acid (HF) are examples of

chemical agents for creating a fuzzy structure on the titanium surface. When etching an

implant surface a transformation occurs from anisotropy to isotropy. In an animal study on

rats (Butz et al. 2006) T-shaped implants were either turned by machining or treated by acid-

etching with HCl and H2SO4 and inserted in rats and evaluated after two and four weeks. It

was concluded that bone integrated to the acid-etched surface was harder and stiffer than bone

integrated to the machined surface. In a study including 97 patients and 432 implants (Khang

et al. 2001) the cumulative implant success rate was 95% for dual-etched implants and 86.7%

for machined implants after 36 months of healing, giving a significant difference related to

surface characteristics. Regardless, no major clinical differences are reported from the

literature when comparing etched surfaces and turned implants (Wennerberg and Albrektsson

2009).

Blasted and etched surfaces

In order to smooth sharp edges after the blasting procedure the implant surface sometimes is

etched with acid. This procedure might also facilitate protein adhesion, which might affect the

early bone-healing process positively. In an animal study (Buser et al. 1991), electropolished,

blasted and etched and HA-coated implants were placed in the metaphyses of the tibia and

femur in miniature pigs. The highest extent of bone-implant contact was observed in

sandblasted and acid treated surfaces after 3 and 6 weeks of healing. It was concluded that the

extent of bone-implant contact was positively correlated with an increasing roughness of the

implant surface. On the other hand, no difference in bone-forming capacity was found

regarding rough (TPS) surfaces, moderately rough (oxidized and blasted + etched) and

minimally rough implant surfaces when compared in dogs after 3 months of loading (Al-

Nawas et al. 2008). In a human histological study by Grassi et al. (2006), microimplants with

18

a sandblasted + etched surface were compared with microimplants with a machined surface 2

months after insertion in human bone. A statistically significant difference concerning bone-

to-implant contact between the two surfaces was found. Microimplants with sandblasted +

etched surfaces had a higher amount of bone in direct contact to the threads. In summary,

sandblasted and etched surfaces exhibit stronger integration in bone compared to machined

surfaces (Wennerberg and Albrektsson 2009).

Oxidized surfaces

Increasing the oxide layer on dental implants might influence the implant incorporation in

bone positively. The process is achieved either with heat or by placing the implant in a

galvanic cell leading to a thickening of the oxide layer on the implant surface. In an animal

study by Choi et al. (2006), the effects of high anodic oxidation voltages on the surface

characteristics of titanium implants and the biological response of rabbit tibia were tested.

Prepared anodized implants were tested in 4 groups with different oxidation voltage (300-550

V). The removal torque values were measured and histomorphometric analysis was done after

1 and 3 months of healing. Oxide layer thickness increased rapidly and pore size also with

voltage up to 500V but decreased at 550 V. Removal torque and bone-to-implant contact

increased with higher voltage compared to 300 and 400 V. In a human study by Shibli et al.

(2007), microimplants with a turned surface and microimplants with oxidized surfaces were

compared. The authors concluded that oxidized implants demonstrated more bone-to-implant

contact than turned surfaces. In general, oxidized implants demonstrate stronger bone

anchorage than machined implants, in animal as well as in human experiments (Wennerberg

and Albrektsson 2009).

Plasma spraying technique (TPS)

In order to create a granular implant surface configuration titanium particles are applied with

a plasma spraying technique. Animal studies have found TPS surface to be better integrated in

bone compared to smoother implants (Gotfredsen et al. 2000, Lee et al. 2004). However, in

clinical studies, increased marginal bone resorption and infection rate have been found around

implants with TPS surfaces. Therefore the use of TPS implants has decreased (Åstrand et al.

2000, Becker et al. 2000).

Electropolished surfaces

Electropolishing is an electrochemical process that removes material from an implant in order

to create a micro-rough surface. Today, studies are sparse concerning the importance of

nanometre scale structures on implant integration in bone. In one clinical study on humans

(Goené et al. 2007) microimplants with dual-etched surfaces were compared with and without

nanometre deposits of CaP. All microimplants were placed in the maxilla. After 8 weeks of

healing the microimplants were removed and examined by scanning electron microscopy. The

bone-to-implant contact was statistically significant higher for the CaP-treated surface. This

could indicate that nanometre structures have a positive impact on early bone healing.

19

Implant follow-up Even though strict criteria have been introduced, most studies still lack information about

implant success rate. Implants can basically be judged as either stable or mobile. A non-

osseointegrated implant should be removed and all remaining implants would then be

classified as survivals. In order to evaluate the remaining dental implants well-defined criteria

for success are important. Consequently, it is important that success criteria are defined and

agreed upon before any follow-up study is performed. In 1986 (Albrektsson et al.) five criteria

for implant success were defined and those have become the most frequently used:

1) An individual, unattached implant is immobile when tested clinically.

2) A radiograph does not demonstrate any evidence of peri-implant radiolucency.

3) Vertical bone loss of at most 1 mm after the first year and 0.2mm annually following

the implant’s first year of service.

4) The individual implant performance can be characterized by an absence of persistent

and/or irreversible signs and symptoms such as pain, infection, neuropathy,

paresthesia or violation of the mandibular canal.

5) Thus, in the context of the four criteria above, a success rate of 85% at the end of a

five-year observation period and an 80% rate at the end of a ten-year period should be

the minimum criteria for success.

Further adjustments have been made in order to make the criteria suitable for individual

implant evaluations. In 1993, implants were classified into four categories: successes,

survivals, unaccounted for and failures in order to individually characterize and evaluate

dental implants (Albrektsson and Zarb 1993).

Back-scattered electron imaging and energy dispersive

spectroscopy Scanning electron microscopy (SEM) is a well known technique used for micro-anatomical

imaging. Scanning an electron beam across a specimen creates high resolution images of

morphology and topography (Sriamornsak and Thirawong 2003). The electrons are usually

generated from a scheelite cathode, transported through condenser lenses and deflection coils

before they reach the sample surface. Back-scattered electrons (BSE) are electrons that are

reflected from the sample. Detectors receive the emitted secondary electrons and provide

information about the sample topography. Occasionally some electrons hit an atomic nucleus

and are repelled back. These primary back-scattered electrons contain compositional

information about the specimen since the high energy signal is proportional to the atomic

number of the element. Areas with heavy elements appear light and areas with light elements

appear dark. Therefore BSE is an excellent method to distinguish contrast between bone and

resin. In general the resin appears black and bone white due to the content of each material

(Slater et al. 2008). Different types of samples can be exploited by SEM such as dried

specimens non-embedded or embedded in resin, frozen-wet tissue or damp-wet tissue.

However, when using BSE it’s advantageous to examine a flat specimen, since the method is

sensitive to deviant topography (Sriamornsak and Thirawong 2003). All non-conductive

specimens are mounted rigidly on an aluminium stub and must be electrically conductive and

grounded in order to prevent electrostatic charge at the surface and image artifacts. Different

techniques of coating non-conductive specimens are used today, for instance gold, platinum,

osmium and carbon/graphite are spread over the specimens in a thin layer before scanning.

Energy dispersive X-ray spectroscopy (EDS) is an analytical technique used to identify

elements present in an object. An electron beam hits an electron in an atom, which gains

energy and jumps to a higher energy level. When it falls back to its original orbital the energy

20

it gained is emitted as an X-ray. The emitted X-rays are detected and measured by an energy

dispersive spectrometer. Since the energy of the x-ray is characteristic for different elements

the composition of a sample can be clarified (Goldstein et al. 2003). A combination of BSE

and EDS has proven to be useful when investigating either the composition of a residual

material or the resorption process (Slater et al. 2008, Tonino et al. 2008).

Bone Tissue Engineering, growth factors and scaffolds Tissue engineering is a relatively new element in the history of biomaterials, which combines

stem cells, therapeutic molecules (growth or differentiating factors) and natural/artificial

scaffolds to replace or improve biological functions. In brief, bone marrow is harvested from

human tissue. The mesenchymal stem cells (MSCs) need to be isolated in order to

differentiate. When a sufficient number have been reached the MSCs are seeded on a

resorbable synthetic scaffold. The scaffold is then inserted in the defective area (Petite et al.

2000).

Tissue engineering has been evaluated in both animal and clinical studies. Attempts to restore

large bone defects have been ineffective (Caplan 1991, Derubeis and Cancedda 2004, Khan et

al. 2008), possibly due to difficulties in transferring laboratory procedures to clinical

applications (Petite et al. 2000).

Stem cells are divided into embryonic stem cells and adult stem cells according to their source.

Embryonic stem cells have the highest level of pluripotency and have unlimited self-renewal,

but ethical issues today limit the use of these cell-types. However, using adult stem cells is

less controversial. They can be harvested from many sources and are immune-privileged.

Adult stem cells have a decreased differentiation capacity and have a limited self-renewal.

Many adult tissues contain stem cells that can be isolated. The most documented tissue being

used for isolation of stem cells is bone marrow, which contains hematopoietic stem cells

(HSCs) and mesenchymal stem cells (MSCs). HSCs generate all types of blood cells and

MSCs can be differentiated into osteoblasts, chondrocytes, adipocytes and myoblasts

(Prockop 1997) in order to regenerate human tissues.

Growth factors play a crucial role in bone tissue engineering. They consist of signalling

molecules/cytokines triggered by many different cell types. By releasing growth factors

MSCs and endothelial cells (EC) can communicate and regulate both angiogenesis and

osteogenesis cell activity (Wozney and Seeherman 2004). These molecules are essential and

play an important role in tissue engineering (Hallman and Thor 2008). Bone morphogenetic

proteins (BMPs) belong to the transforming growth factor (TGF) beta family and are the most

researched osteoinductive bone factors. BMPs are regarded as the most important regulators

of bone regeneration (Termaat et al. 2005, Canalis 2009). Multiple growth factors with

different properties are expressed during bone formation, such as BMPs, fibroblast growth

factor (FGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF),

transforming growth factor β (TGF- β) and vascular epithelium growth factors (VEGF)

(Jadlowiec et al. 2003).

Skeletal tissue engineering requires a scaffold in combination with different osteoprogenitor

cells and osteoinductive growth factors. The scaffold itself must fulfil primary functions for a

successful treatment of skeletal defects. It must be biodegradable, biocompatible, promote

cellular interactions and tissue development, provide temporary mechanical load-bearing

within the tissue defect and also have suitable physical properties (Nair and Laurencin 2007;

Salgado et al. 2004).

Recently, several different new synthetic bone-substitute materials have been produced and

introduced. Most common are ceramics, polymers and their combination. Ceramics such as

hydroxyapatite Ca10(PO4)6(OH)2 and tricalcium phosphate Ca3(PO4)2 are some of the most

used bone substitutes due to their superior osteoconductivity and bone-bonding ability.

21

Nevertheless, absence of osteoinductiveness and limits to repairing large segmental bone loss

(Marcacci 2007) have made it attractive to find more degradable scaffolds for tissue

engineering such as polymers. Both polymers of natural origin, such as collagen, chitosan,

fibrinogen, starch, hyaluronic acid and poly(hydroxybutyrate), and synthetic origin, such as

poly(lactic acid), poly(glycolic acid) and poly(ε-caprolactone) have been developed and

investigated for tissue engineering applications (Griffith 2002, Salgado et al. 2004). Polymers,

in contrast to hydroxyapatite and tricalcium phosphate, are more flexible in design and

composition. The biodegradable polymers can be produced in special patterns enabling

manufacturing of polymers with different characteristics, such as porosity, degradation rates

and interconnectivity (Nair and Laurencin 2007, Porter et al. 2009). It has been shown in a

series of both in vitro and in vivo experiments that copolymers can be promising as scaffolds

for bone tissue engineering (Dånmark et al. 2010, Idris et al. 2010, Schander et al. 2010).

At present, stem cell-based bone tissue engineering is a promising method for many fields of

interest, for example evaluating surface attachment and cell proliferation on bone substitutes,

but further research is necessary and on-going.

22

AIMS

General aim

The overall aim of the present investigation was to study the graft healing and tissue

responses to biphasic calcium phosphate after sinus floor augmentation.

Specific aims

a) Histological and histomorphometrical evaluation of microimplants placed in DBB or

BCP after 8 months of graft healing

b) Evaluation of the resorption of BCP and DBB by comparing the appearance of the

remaining particles after 8 months of graft healing and determine whether any changes

in the Ca/P ratio could be detected due to the dissolution of the β-TCP component

during the healing process

c) Evaluation of Straumann®

SLActive dental implants placed in either BCP or DBB after

1 year and 3 years of functional loading

d) Histological and histomorphometrical evaluation of BCP and DBB after 36 months of

graft healing

e) Evaluation of the response of human osteoblast-like cells (HOB) to nDP-modified and

un-modified DBB and BCP scaffolds

23

MATERIALS AND METHODS Patients and study outlines

Paper I, II, III and IV

In this randomized and controlled study, bilateral maxillary sinus floor augmentation (MSFA)

was performed in 11 patients with severe atrophy in the posterior maxilla (Cawood & Howell

class V and VI) using biphasic calcium phosphate (BoneCeramic®) at one side and

deproteinized bovine bone (Bio-Oss®

) at the contralateral side acting as control.

Microimplants were installed in conjunction with the sinus augmentation. After 8 months of

graft healing, the microimplants with a surrounding bone core were removed with a trephine

burr. Histomorphometric and elemental analysis were performed. Simultaneously, a total of

62 Straumann®

SLActive implants were installed with an average healing time of 118 days

(range, 97-210 days) before the prosthesis was initiated.

In Paper I, comparison of bone formation around microimplants placed in BCP and DBB was

carried out. In Paper II, the composition of residual graft material of BCP and DBB and

surrounding bone was analysed by scanning electron microscopy and energy dispersive X-ray

spectroscopy. In Paper III, implant success rate was evaluated after one year of functional

loading. In paper IV implant stability was estimated after three years of loading. Radiographic

evaluation and resonance frequency analysis (RFA) were performed and implant success rate

as well as three-year implant survival rate was calculated. The health of the maxillary sinuses

was also examined by means of CT three years after the grafting procedure. In addition, after

three years of graft healing 18 new biopsies were harvested in 9 patients (2 patients did not

want to participate) for light microscopic analysis and morphometry (Table 1).

Paper V

The aim of the present in vitro study was to evaluate the response of human osteoblast-like

cells (HOB) to nano-crystalline-diamond-particle-modified (nDP-modified) and un-modified

(control) deproteinized bovine bone (DBB) and biphasic calcium phosphate (BCP) scaffolds.

nDP-modification of DBB and BCP- particles was carried out through different steps of

preparation including grinding and ultrasonic technique. In each experiment, 100 mg

materials from each of the 4 groups (nDP-modified DBB, nDP-modified BCP, un-modified

DBB and un-modified BCP) were plated into a 48 well cell culture plate and 200.000

cells/well were seeded onto the materials. Scanning electron microscopy (SEM) was carried

out after 24 hours and 3 days. Real time-polymerase chain reaction (PCR) was carried out

after 3 days, 1 week and 2 weeks of incubation. The following osteoblast differentiation

markers were analyzed; alkaline phosphatase (ALP), osteocalcin (OC), bone morphogenetic

protein type 2 (BMP-2) and integrin alpha 10 (ITGA 10). The general tendency of DBB and

BCP was compared.

24

Patients Sinus

lifts

Microimplant

biopsies

Elementary

analysis

3-years

biopsies

Dental

implants

Dental

implants

3-years

11 22 22 12 18 62 60

Table 1. Study material used in papers I-IV.

Presurgical examination, inclusion and exclusion criteria

Prior to surgery all patients were clinically and radiographically examined. The radiographic

examinations were based on tomograms (Scanora, Soredex Orion Corp Helsinki Finland)

(Figure 1). The maxillary bone was examined with respect to the shape and volume of the

residual alveolar process and regional anatomy.

Figure 1. Pre-surgical panoramic radiograph of a study patient, showing less

than 5mm (arrows) of residual bone in the floor of the maxillary sinus.

Inclusion criteria

Patients were included in this study if no systemic or local contraindications were encountered

and if they had a need for bilateral sinus augmentation. The patients should have less than

5mm of residual bone in the floor of the maxillary sinus and a crestal width of at least 4mm.

Exclusion criteria

Patients were excluded if they had any severe disease such as: uncontrolled diabetes, active

sinus infection, uncontrolled periodontal decease or history of head and neck radiation or

25

chemotheraphy treatment. Furthermore, patients were excluded if they had physical problems

that would prevent long-term treatment or were smoking more than 10 cigarettes per day.

Ethics committee approval

Study I, II, III and IV were approved by the regional ethical research committee at the

University Hospital in Uppsala, Sweden. All patients were given written information about

the study, and their consent was registered in their charts. Study V was approved by the

ethical authorities at the University of Bergen, Norway.

Implants

Microimplants

A total of 22 specially designed screw-type microimplants (Figure 2) with the Straumann®

SLA surface (sandblasted, large-grit, acid-etched) were used in study I and II. The

microimplants were 10mm long with a threaded body that was 2mm in diameter and a slotted

head (2mm high).

Figure 2. The titanium microimplant used in the study.

Routine implants

A total of 62 placed Straumann® SLActive implants, were followed in studies III and IV.

SLActive®

implants have a hydrophilic surface and a different nano-roughness than SLA®

surfaces. All implants were 8 to 12mm in length and 3.3 to 4.1mm in width. Twenty-four

implants were placed in sites grafted with biphasic calcium phosphate (BCP), 23 implants in

sites grafted with deproteinized bovine bone (DBB) and 15 implants were placed in residual

bone close to the augmented areas.

26

Grafting materials

Biphasic calcium phosphate (BCP)

The BCP that was used in the present thesis for maxillary sinus floor augmentation is based

on a balance between a more stable phase (HA) Ca10(PO4)6(OH)2 and a more soluble phase

(β-TCP) Ca3(PO4)2. Straumann BoneCeramic® is a fully synthetic bone substitute and consists

of 60% HA and 40% β-TCP in a hard sintered mixture. The specific morphology of

BoneCeramic®

enables vascularization, osteoblast migration and bone deposition. Straumann

BoneCeramic®

is 90% porous with interconnected pores of 100-500 microns in diameter. The

particle size varies between 250-1000µm.

Deproteinized bovine bone

Bio-Oss® (Geistlich, Pharmaceutical, Wolhusen, Switzerland) is derived from bovine bone

where all organic material has been removed in a strong alkaline solution (> pH 13 for 4

hours), and sterilized and later heated for 15 hours in 300°C. Bio-Oss has a natural porous

system and the morphological and chemical properties are similar to human bone. The

particle size that was used in this study varied between 250 and 1000 µm.

Surgery

Sinus surgery

As a prophylactic measure, all patients received 1g penicillin-V (Kåvepenin; Astra®)

preoperatively and three times daily for 7 days. Sinus augmentation was performed bilaterally

in all patients. In brief, after a crestal incision and two vertical releasing incisions, a

mucoperiosteal flap was elevated and reflected laterally. A window approximately 20mm

wide and 10mm high was outlined with a bur, and the schneiderian membrane was elevated

together with the bone window inside the sinus cavity (Figure 3). Care was taken not to

lacerate the membrane during the elevation procedure. Augmentation was performed with

BCP in one side of the maxillary sinus floor and with DBB acting as a control on the other

side, according to a randomization procedure (Figure 4). In the BCP group 0.5 to 1.5g (mean,

1.0g) of the material was used for each site, compared to 0.5 to 1g (mean 0.8g) of DBB. In

order to inhibit in-growth of soft tissue into the grafted area, a collagenous membrane

(BioGide®

, Geistlich Pharma) was placed on the lateral wall of the maxillary sinuses. The oral

mucosa was then sutured with resorbable sutures (Figure 5).

Figure 3. Clinical view of surgical entry of the left maxillary sinus.

27

Figure 4. Clinical view showing completed augmentation of

the left maxillary sinus floor with BCP. A microimplant is

visible (arrow).

Figure 5. Clinical view of the sutured oral mucosa.

Placements of microimplants

Simultaneously, with the augmentation procedure, the microimplant sites were prepared

vertically in the region of the second premolar using a 2mm-wide drill. All microimplants

were placed through the residual bone, penetrating the augmented areas and attached using a

small screwdriver until the head reached the surface of the alveolar crest (Figure 6).

28

Figure 6. Panoramic radiograph showing bilateral maxillary sinus

floor grafts after 8 months of healing. On the left side a microimplant

(arrow) is placed in DBB and the right side a microimplant (arrow)

is placed in BCP.

Implant placement

After 8 months of graft healing ordinary dental implants were placed non-submerged and left

to heal for an average healing time of 118 days (range; 97 to 210 days).

Prosthetic procedures

The prosthetic procedure involved removal of covering screws, placement of abutments,

providing fixed bridges and removal of the bridges after a time period of one and three years

of functional loading in order to evaluate the individual implants.

Follow-up

Clinical follow-up was performed at the time of the abutment connection and bridge

connection, as well as after one and three years of functional loading (Papers III and IV), with

the bridge removed before recordings. After three years of functional loading, resonance

frequency analysis (RFA) of the implants was carried out (Paper IV).

Marginal bone resorption

After 1 year of functional loading and after 3 years of functional loading intraoral radiographs

were obtained using either analog sensors (Kodak Ektaspeed Plus; Eastman Kodak) or digital

Schick sensors (Schick technologies, Long Island City USA). The marginal bone level in

relation to the implant shoulder was measured on the left and the right side of each implant on

radiographs taken when the fixed prosthetic constructions were ready for hand out, after 1

year of functional loading and after 3 years of functional loading. All measurements were

carried out using either a loupe (Peak Scale Loupe x 7 measurement scale) or a digital ruler in

Schick images (Paper III and IV).

Graft dimensional changes

To find out if there were any dimensional changes due to resorption of BCP and DBB over

time, measurements were carried out (Paper III). Grafted sinus height (GSH), defined as the

distance from the intraoral marginal bone to the highest point of the grafted area, were

measured both at baseline and after 1 year of loading from either conventional panoramic X-

ray (Kodak Lanex medium; Kodak, Rochester, NY) or digital X-ray panorama (Schick

29

technologies, Long Island City USA). In all measurements the magnification on conventional

panoramic films was taken into consideration (panoramic films x 1.3). All measurements

were made twice.

Health of the maxillary sinuses

After 3 years of functional loading, at the time for clinical recording of implant stability, cone

beam computed tomography (CBCT) (I-CAT®

Cone Beam 3-D Imaging System, Imaging

Sciences International Inc, Hatfield, PA, USA) was carried out in order to examine the health

of the grafted sinuses (Paper IV). The individual maxillary sinus was classified as unhealthy if

any mucosal hypertrophy or fluid was found. Two patients had cemented fixed bridges due to

difficulties with bridge attachment and therefore were excluded in this part of the study.

Resonance frequency analysis

After three years of functional loading, all fixed constructions were removed in 9 of the initial

11 patients and each fixture was evaluated for implant stability by means of resonance

frequency analysis (RFA). A magnetic resonance frequency analyser, Ostell™ equipment

(Integration Diagnostic AB, Gothenburg, Sweden) was used for the purpose (Paper IV).

Resonance frequency values were recorded as quantitative implant stability quotient (ISQ), on

a scale from +1 to +100 where one is the lowest degree of stability and 100 the highest. An

ISQ value below 50 increases the risk of disintegration. A mean ISQ value was calculated for

each implant based on two measurements of each implant. In brief, a transducer was attached

to the implant abutments (Figure 7). The probe of the wireless resonance frequency analyser

was held perpendicular to the alveolar crest and the ISQ value was presented on the screen of

the analyzer (Figure 8). After the measurements, the prosthesis was re-attached.

Figure 7. A transducer was attached to the implant abutments

before digital recording.

30

Figure 8. The magnetic resonance frequency analyser, OstellTM

equipment used in the study.

Histology

Twenty-two microimplants were retrieved with a surrounding bone core after 230 days

(range; 210-250 days) of graft healing (Paper I). After 3 years (range; 35-37 months) of graft

healing, eighteen additional biopsies were retrieved using a trephine bur (Paper IV). All

specimens were fixed by immersion in 4% buffered formalin solution, dehydrated in a graded

series of ethanols (70-100%) and embedded in plastic resin (Technovit A 7210VCL (Paper I)

and Technovit 7200VCL (Paper IV); Kulzer & Co, Hanau, Germany). After the retrieval and

resin embedding of the microimplants, the blocks were divided. One half of the block was

used for preparing sections for histology (Paper I) and the other half was used for electron

microscopy and elemental analysis (Paper II). Sections were cut and ground to a thickness of

approximately 10µm by means of cutting and grinding equipment (Exakt Apparatbau,

Norderstedt, Germany). One central section was prepared from each biopsy. The ground

sections were stained with a mixture of toluidine blue and 1% pyronin-G. Examination,

photography and morphometric measurements were made using a Leitz Orthoplan

microscope equipped with a Microvid morphometric system (Ernst Leitz Wetzlar, Wetzlar,

Germany) connected to an IBM PC (Paper I). The morphometric measurements comprised

measurements of the percentage of implant surface in direct contact with bone, the percentage

of bone area occupying the threads of the microimplants, the percentage of bone, biomaterial

and soft tissue occupying an area inside and outside every two threads that could be measured

and the percentage of graft particles in contact with newly formed bone. In Paper IV,

qualitative and quantitative analyses were carried out using a Nikon Eclipse 80I light

microscope (Belmont, CA, USA) and a Leitz Aristoplan light microscope (Ernst Leitz GmbH,

Wetzlar, Germany) with a Leitz Microvid Morphometric system connected to a personal

computer. The percentage of mineralized bone, residual graft materials and fibrous connective

tissue in the 3-years biopsies of BCP and DBB and the percentage of particles in contact with

newly formed bone were calculated. In order to standardize the analyses a region of interest

(ROI) was chosen in the area where graft material was present using a square grid (6.67mm2).

The same person performed all the measurements on both occasions.

31

Electron microscopy and elemental analysis

The unstained sections of the embedded microimplants were used for electron microscopy

and elemental analysis (Paper II). The blocks were polished using a manual grinder with 800

grit silicone carbide paper, mounted on an aluminium stub and also carbon coated (Polaron

sputter coater). Electron microscopy and analysis were carried out at the Center for Electron

Microscopy, University of Birmingham, UK. Samples were examined using back-scattered

electron imaging, using a field emission environmental scanning electron microscope (Philips

XL 30 FEG ESEM) operating in high vacuum mode at a working distance of 10mm and an

accelerating voltage of 10-15kV. In order to evaluate the elemental composition in graft

material and adjacent bone in 12 specimens (6 DBB and 6 BCP were used for paired

analysis), energy dispersive spectroscopy (EDS) was carried out on the same instrument fitted

with an Oxford INCA 300 EDS System. Points were analysed at 4-6 sites of interest for each

specimen. To investigate any changes in composition at the junctions between bone and graft

material, line scans were carried out to compare the composition along a line spanning a

junction between bone and biomaterial.

Surface modification of biphasic calcium phosphate and deproteinized bovine bone

particles

Nano-crystalline-diamond-particle (nDP) modification of DBB and BCP particles was carried

out through different steps of preparation including grinding and ultrasonic technique (Paper

V). Human alveolar bone specimens were retrieved from patients undergoing routine oral

surgery at the Department of Maxillofacial Surgery, Haukeland University Hospital in Bergen.

Human osteoblast-like cells were harvested and characterized using staining for ALP activity

using Naphthol AS-TR phosphate and Fast red violet B salt (Sigma Aldrich, USA).

Cells from passages 3-5 were expanded in culture and used for the experiments (Figure 9).

Cells were harvested from two different donors. In each experiment, 100 mg materials from

each of the 4 groups (nDP-modified DBB, nDP-modified BCP, un-modified DBB and un-

modified BCP) were plated into a 48 well cell culture plate and 200.000 cells/well were

seeded onto the materials. Scanning electron microscopy (SEM) was carried out after 24

hours and 3 days. Real time-polymerase chain reaction (PCR) was carried out after 3 days, 1

week and 2 weeks of incubation. The following osteoblast differentiation markers were

analyzed: alkaline phosphatase (ALP), osteocalcin (OC), bone morphogenetic protein type 2

(BMP-2) and integrin alpha 10 (ITGA 10).

Figure 9. Morphology of human osteoblast-like cells after 3 days in

expansion medium (scale bar = 100µm).

32

Statistics

In Paper I, Wilcoxon’s Signed Rank test was used for paired morphometrical analysis.

According to a power calculation 11 patients were enough to detect a difference of = 15

between the test and control group (Table 2).

In Paper II the Ca/P (atomic %) ratios were calculated from the point analysis data from

patients augmented with either BCP or DBB. The data were classified according to location.

Sets of data were averaged and analysed using Wilcoxon’s Signed Rank test for paired data.

In Paper III, differences between baseline and 1-year follow up regarding marginal bone level

between implants placed in BCP and DBB were analysed using binomial tests. One-way

analysis of variance (ANOVA) was used to analyse differences between clinical findings

regarding implants placed in BCP, DBB or in residual bone and paired t-tests were used to

analyse clinical differences between BCP and DBB. In order to illustrate whether there were

any differences concerning marginal bone resorption between implants placed in DBB or

BCP, with the patient as “n” instead of the implants, a dependent t-test was carried out.

Analysis of the grafted sinus height (GSH) was performed using a paired t-test.

In Paper IV, differences between mean marginal bone levels for implants placed in BCP,

DBB or in residual bone and implant survival/success rate were calculated using one-way

ANOVA. To calculate differences between baseline and 3 years of loading for each group a t-

test was carried out. Comparison between baseline, 1 year of loading and 3 years of loading

regarding statistical differences between implants placed in BCP or DBB was made using a

paired t-test. One-way ANOVA was used to analyse SBI, PLI, BPI and PPD around the

implants placed in BCP, DBB or in residual bone. To evaluate differences concerning RFA

between implants placed in BCP, DBB or in residual bone one-way ANOVA was also carried

out.

In Paper V, all values of PCR results were presented as mean ± SD. Both normality and equal

values tests were performed and passed suggesting that a parametric test could be used.

Ordinary t-testing was performed for comparison of two groups.

In all Papers, the level of significance was set to p ≤ 0.05.

Table 2. The table shows the sample size for the 2-sided paired t-test (Paper I) with α=0.05 and a power of

80% for the various mean differences and standard deviations.

= 5 = 10 = 15 = 20 = 25 = 30 = 35

= 10 34 10 6 5 4 4 3

= 15 73 20 10 7 6 5 4

= 20 128 34 16 10 8 6 5

33

RESULTS

Histological, elemental, clinical and radiographic outcome (Papers I-IV)

Histological results (Papers I and IV)

After 8 months of graft healing all specimens contained varying amounts of vascularised

fibrous connective tissue, DBB and BCP particles and various amounts of woven and lamellar

bone (Paper I) (Figure 10). No obvious signs of resorption were noticed for the BCP and DBB

particles (Figures 11 and 12). Bone-to-implant contact (BIC) in the BCP group compared to

the DBB group was 64.6 ± 9.0% versus 55.0 ± 16.0%; p=.07 (not significant: NS). The

corresponding values for the area of newly formed bone in the biopsies were 41.1 ± 9.8% and

41.6 ± 14.0% for BCP and DBB, respectively (NS). There was a significantly higher number

of DBB particles in contact with newly formed bone compared to BCP (mean 87.9 ± 18.2%

versus 53.9 ± 26.1%; p = 0.007). The amount of remaining graft occupying the biopsy after 8

months was 12% for DBB particles and 10.8% for BCP. The difference was not significant. A

non significant difference between the two biomaterials for area of newly formed bone and

soft tissue was also found. No pathologic signs of inflammatory activity were noticed. After 3

years of graft healing (Paper IV) (Figures 13 and 14) the areas of bone in the biopsies were

28.6% ±14.3% and 31.7% ± 18.0% for BCP and DBB respectively (NS). The amount of soft

tissue components in the groups were 51.0% ± 15.0% for BCP and 44.3% ± 10.5% for DBB

(NS) (Figure 15).

Graft particles in contact with bone in the BCP group compared with the DBB group were

37.8% ±10.9% versus 44.4% ± 12.1% (NS). BCP particles were found to be under different

levels of dissolution. The dissolution was mostly observed on the edges of the BCP particles

but in some cases the entire particle was undergoing dissolution (Figure 16). However, there

were no statistically significant differences between the areas of BCP and DBB, which were.

20.4% ± 7.5% and 24.0 ± 13.5% respectively. After 3 years of graft healing, BCP samples

demonstrated a greater number of inflammatory cells compared to the DBB samples.

Figure 10. Results from the histomorphometric analysis after

8 months of graft healing showing the mean percentages of

new bone in contact with titanium, bone area inside threads,

and any particles with bone contact inside and outside threads

34

(P = .007).

Figure 11. Light micrograph after 8 months of graft

healing showing BCP (BCP) particles partly in contact

with newly formed bone (B) and fibrous connective

tissue (C). The implant is in contact with bone that

bridges the BCP particle (arrow).

Figure 12. Light micrograph after 8 months of graft

healing showing DBB particles (DBB) to be well

incorporated with newly formed bone (B) and fibrous

connective tissue (C). The implant is in contact with bone

that bridges the DBB particle (arrow).

BCP

BCP

C

C

B

B

DBB

DBB DBB

C

C

B

B

BCP

35

Figure 13. Light micrograph after 3 years of graft healing showing BCP

particles (BCP) to be well incorporated with newly formed bone (B)

and fibrous connective tissue (C).

Figure 14. Light micrograph after 3 years of graft healing showing

DBB particles (DBB) to be well incorporated with newly formed bone

(B) and fibrous connective tissue (C).

36

Figure 15. Results from the histomorphometrical

analysis 3 years after augmentation, showing the mean

percentages of remaining graft particles, bone, soft

tissue (fibrous connective tissue (FCT)) and particles in

contact with bone within the ROI. There were no

statistically significant differences (P > .05) between the

groups for any parameters. BC = BoneCeramic,

DBB = BioOss.

Figure 16. Light micrograph showing BoneCeramic particles (BCP)

under different levels of dissolution both in contact with bone (B),

including two immature osteocytes visible in the lacunae (OL), and in

contact with soft tissue (C). The edge of the particle has bone-like

material incorporated in the surface (arrows).

37

Results of elemental analysis (Paper II)

The bone-graft substitute interface for BCP showed evidence of superficial disintegration of

particles into individual grains. Median Ca/P ratios (atomic %), determined from >200

discreet sites within residual graft particles and adjacent bone, were: DBB: 1.61 (CI 1.59-

1.64); BCP: 1.5 (CI 1.45-1.52); DBB-augmented bone: 1.62 (CI 1.59-1.66); BCP-augmented

bone: 1.52 (CI 1.47- 1.55); P = 0.028 for DBB versus BCP and DBB- versus BCP-augmented

bone) (Figure 17). The reduction in Ca/P ratio for BCP over the healing period is consistent

with the dissolution of β-TCP and reprecipitation on the surface of calcium-deficient

hydroxyapatite.

Figure 17. Boxplots showing Ca/P ratios determined from point

analysis data from graft particles, adjacent bone and at the

bone-material interface. The data are from 6 patients except for

the interface data which are from only 3.

Clinical results (Papers III, IV)

One implant placed in BCP was lost after 3 months of functional loading due to infection, and

one implant placed in DBB was lost only a few weeks after insertion due to lack of initial

stability. After a mean of 36 months (range; 36 to 37 months) of functional loading the overall

implant survival rate was 96.8%. No significant difference was found between implants

placed in BCP, DBB and residual bone.

The mean PPD values were 2.6mm ± 0.8mm, 2.3mm ± 0.7mm and 2.6mm ± 1.2mm for BCP,

DBB and residual bone respectively. No statistically significant differences were observed for

plaque index (PLI), bleeding on probing index (BPI), sulcus bleeding index (SBI) or probing

pocket depth (PPD) irrespective of group. The success rate for implants placed in BCP, DBB

and residual bone were 91.7%, 95.7% and 86.7% respectively.

The mean RFA values differed between the groups (Paper IV). In total, 52 implants were

measured, nineteen placed in BCP, 18 in DBB and 15 in residual bone. The mean ISQ values

were 68.5 ± 5.1 (range; 62 to 79), 70.4 ± 4.3 (range; 59 to 77) and 64.2 ± 7.1 (range; 51 to 80)

38

respectively. There was a statistically significant difference between implants placed in DBB

and residual bone, (p = 0.008). The other comparisons were non-significant (Figure 18).

Figure 18. Plot showing resonance frequency analysis of implants

placed in BCP (BoneCeramic), DBB (Bio-Oss) and residual bone

(Residual). Bars indicate 95% confidence intervals (CI).

Radiographical results (Papers III, IV)

The mean marginal bone loss from baseline to 1 year of loading (Paper III) was 0.29 ± 0.10

mm and 0.43 ± 0.20 mm for implants placed in DBB and BCP, respectively. Implants placed

in residual bone had a mean marginal bone loss from baseline to 1 year of loading of 0.52 ±

0.27. No statistically significant difference between baseline and 1 year of loading was found

according to marginal bone levels around the implants placed in DBB, BCP and residual bone.

The amount of graft resorption for DBB compared to BCP was 4% and 4.5% after 1 year of

loading (NS).

The mean marginal bone loss from baseline to 3 years of functional loading differed between

implants placed in BCP, DBB and residual bone (Paper IV) (Figure 19). The mean marginal

bone loss was 1.32 ± 1.10mm for implants placed in BCP, 1.10 ± 0.78mm for implants placed

in DBB and 2.10 ± 1.34mm for implants placed in residual bone. No statistical differences in

marginal bone loss could be observed between the implants placed in BCP or DBB. Between

baseline and 3-years of loading there were statistically significant differences concerning

marginal bone level comparing dental implants placed in either BCP or DBB and residual

bone respectively. Two implants placed in residual bone and 1 implant placed in BCP had a

mean marginal bone loss exceeding 2.4mm leading to a calculated success rate of 91.7%,

95.7% and 86.7% for implants placed in BCP, DBB and residual bone respectively.

Four of 18 examined sinuses (two in each group) showed moderate signs of mucosal

hypertrophy (22.2%) and 12 were judged healthy (77.8%). No patients had any clinical

symptoms.

39

Figure 19. Plot showing the mean implant marginal bone loss

from baseline to 3 years of loading for implants placed in

DBB (Bio-Oss), BCP (BoneCeramic) and residual bone

(Residual). A statistically significant difference between DBB

and residual bone concerning marginal bone loss after 3 years

of loading was found (p=.028). No statistical difference could

be seen regarding marginal bone loss around the implants

placed in BCP or DBB. Bars indicate 95% confidence

intervals (CI).

SEM- and PCR analysis (Paper V)

Our preliminary analysis showed that findings from donor 1 were similar to donor 2. Data

from donor 2 is not presented. The general tendency of DBB and BCP was compared. The

gene expressions between DBB versus BCP were not compared statistically due to different

origin, pore size and porosity. Cellular responses were evaluated in terms of attachment and

differentiation. SEM after 24 hours and 3 days of incubation disclosed similar cell attachment

and spreading for nDP-modified and non-modified DBB and BCP particles. The human

osteoblast-like cells were mainly flat with no specific orientation and could sometimes be

observed aligning themselves with fine irregularities (Fig 20).

Real-time PCR revealed significant up-regulation of mRNA expression of ALP and OC in

HOB grown on nDP-modified DBB and BCP-particles after 1 and 2 weeks compared to non-

modified particles. A significant down-regulation of BMP-2 on nDP-modified DBB (Fig 21a)

and a significant up-regulation of BMP-2 on nDP-modified BCP (Fig 21b) were observed for

HOB in relation to un-modified particles. Cell adhesion marker ITGA 10 showed significant

down-regulated in the mRNA level for both nDP-modified groups after 2 weeks of incubation

(nDP-BCP (p<.01) and nDP-DBB (p<.05) compared to the non-modified materials.

40

Figure 20. Scanning electron micrograph demonstrating an

HOB cell 3 days after attachment on a nDP-modified BCP

particle. The cells could sometimes be seen aligning

themselves with fine irregularities (arrows) for cellular

bridging.

41

Figure 21a. Relative mRNA expressions of osteogeneic and cell attachment

markers by real time PCR after 2 weeks of HOB cultivation on DBB (Bio-Oss)

particles.

Figure 21b. Relative mRNA expressions of osteogeneic and cell attachment markers

by real time PCR after 2 weeks of HOB cultivation on BCP (Strau) particles.

42

DISCUSSION

Paper I

Comments on aim, materials and methods

The aim of this clinical study was to histologically evaluate bone formation around

microimplants with a sandblasted and acid-etched surface and the bone tissue responses to

BCP compared with DBB when used for maxillary sinus floor augmentation.

The idea of using biphasic calcium phosphate for augmentation in implant surgery is based on

the fact that the material consists of a sintered mixture of HA and β-TCP. The β-TCP part will

dissolve over time while the HA part will maintain the supportive structure for bone

formation (Jensen et al. 2006). DBB is a well documented bone substitute and has been

suggested as the standard of care for augmentation of the maxillary sinus (Esposito et al.

2006). Therefore DBB was chosen as the control material to evaluate BCP.

During the fragmentation of β-TCP, calcium and phosphate ions are released, which in theory

are suggested to stimulate bone formation (Daculsi 1998). The specially designed

microimplants used in this study had an SLA® surface. In order to optimize this study it

would have been desirable to use SLActive®

surface modification on both microimplants and

ordinary implants. Due to technical difficulties this was not feasible. The microimplants were

placed vertically thus allowing evaluation of the tissue composition exactly where the bone is

needed to support the implants. However, this technique may jeopardize the ordinary implant

placement. An alternative technique that has been evaluated (Hallman et al. 2002, Artzi et al

2005) is biopsies harvested from a horizontal direction from buccal to palatal direction in the

region of the bony window. This technique allows the possibility of harvesting biopsies

without interfering with the ordinary implant sites. Nevertheless, this technique only evaluates

the tissue composition of the augmented sinus in the deepest region. In some cases there were

difficulties in retrieving the microimplants. In a few cases the apical part of the bone core had

fractured due to the direction of the trephine bur.

To avoid in-growth of soft tissue in the grafted areas and movement of the grafted particles, a

collagenous membrane (BioGide®

) was used. A BioGide® membrane has 2 functional layers,

one compact layer that is cell occlusive and fulfills the barrier function and a porous layer that

allows tissue integration. The membrane is resorbable and has hydrophilic properties, which

enable its self-adherence to different structures. Considering earlier scientific data regarding

bone formation and implant survival, membranes should be used for sinus elevation

procedures (Tarnow et al. 2000, Wallace et al. 2005).

Comments on results

In this 8 month histological study of microimplants placed in either BCP or DBB it was

concluded that the SLA-microimplants were well osseointegrated in both materials and no

complications were reported during the healing period. Hence, it’s obvious that

microimplants can be placed in grafted areas and retrieved without jeopardizing later regular

implant treatment even though in some cases the initial stability of the microimplant stability

was not optimal.

During the specimen processing the ground sections were stained with toluidine blue and 1%

pyronine-G. The staining procedure might present some problems, such as possible

differences in the resulting colour for different samples and the presence of cutting artifacts,

which sometimes are visible as stripes.

43

The result from the morphometrical analysis revealed no statistical differences concerning

mean percentage of newly formed bone, bone graft material and amount of soft tissue

between BCP and DBB. Since the retrieved biopsies contained microimplants the

osteoconductive properties of the titanium must be considered. Furthermore, additional

analysis revealed an interesting incidental finding. The bone-to-graft contact was significantly

higher for DBB than BCP (P=.007). Bone formation in the BCP group mainly occurred

between the BCP particles and close to the microimplant surfaces, compared to the DBB

group, where bone formation was seen primarily around the DBB particles. Since there was a

non-significantly higher bone-to-implant contact for the BCP group compared to DBB this

may indicate that titanium itself is more attractive for bone building cells than the BCP graft

material. The result is in accordance with a similar histological study (Cordaro et al. 2008).

The reasons for this difference are not clear. One contributing factor might be the

fragmentation of the BCP particles into small grains, which might affect the attraction for

bone-forming cells. Another explanation for the lower bone-to-graft contact for BCP might be

that the bone-forming cells are influenced negatively by the surface characteristics of the

particle. This corresponds with other results from in vitro and experimental studies using BCP

(Buser et al. 1998, Jensen et al. 2006, John et al. 2003, Rice et al. 2003, Wang et al. 2004,

Curran et al. 2005). Nevertheless, since the amount of newly formed bone in the two groups

was not statistically different, one can speculate upon the clinical relevance of the lower bone-

to-graft contact for BCP particles. The findings from our study raised the question for an

alternative study trying to find answers to; why the bone-to-graft contact was significantly

lower for BCP than DBB since there was no significant difference in bone-to-implant contact

(Paper V).

Paper II

Comments on aim, materials and methods

The purpose of this study was to investigate the resorption pattern of BCP particles in

comparison with DBB particles by comparing the appearance of the remaining particles after

8 months of graft healing using back-scattered electron imaging (BSE) and energy dispersive

spectroscopy (EDS). A further purpose was to observe the interface between bone and graft

material to determine whether any changes could be detected due to the dissolution of the β-

TCP component during the healing process.

Back-scattered electron imaging is an excellent method to distinguish graft from human tissue.

In BSE images the newly formed bone appear darker gray due to the marrow, collagen, fat

and non-collagenous protein content compared to the graft particles that appear white gray

because of non or low organic content. Furthermore, the method provides information about

surfaces that would be impossible to study in an ordinary light microscope.

Energy dispersive spectroscopy provides information about the chemical composition in a

subject and changes in the chemical composition over time can also be assayed. Nevertheless,

BSE and EDS used together is an easily affected method that requires technical support and

continuous calibration, which must be considered. In order to obtain adequate results biopsies

used for BSE and EDS should be harvested from representative areas and handled under

certain circumstances, by means of specific cutting, grinding and coating procedures. In the

present study EDS paired data were presented from 6 patients due to some of the biopsies

required further preparation before analysis could be carried out.

44

Comments on results

EDS analysis of the samples showed that the Ca/P ratios of retrieved BCP particles were

lower than for the starting material and lower in retrieved biomaterial and bone adjacent to

BCP in comparison with residual DBB particles and bone adjacent to them. One explanation

for the change in Ca/P ratio for BCP might be that the β-TCP part is being replaced by Ca-

deficient HA with a Ca/P ratio of approximately 1.5. In a study by Slater et al. (2008) the

composition of residual calcium sulphate (CaS) used as bone graft substitute in the maxillary

sinus of human subjects, was investigated. The results from this study revealed that CaS

resorption is accompanied by CaP and calcium-deficient HA precipitation, which may be a an

essential step in bone bonding and contribution to the biocompatibility of this bone graft

substitute material. In the present study, β-TCP gradually was replaced by Ca-deficient HA

which could mean that the material actually becomes more stable and remains for a long time

in the tissues. A possible side effect from the resorption phase might be that the initial smooth

BCP surface turns rougher during the years. One can speculate upon whether the initial BCP

surface might be to fine-textured for protein adsorption and adhesion of bone building cells.

Furthermore, one reason for bone adjacent to BCP also having a lower Ca/P ratio than bone

adjacent to DBB could be because it has incorporated some small grains of BCP from the

breaking up of the material during the healing process. This study indicated that BCP particles

might not have the optimal properties for initial bone healing. In order to study the resorption

pattern over time, an interesting complement to these results would be to perform BSE and

EDS on the biopsies harvested after 3 years of graft healing.

Papers III and IV

Comments on aim, materials and methods

The purpose of the 1 and 3 year clinical studies was to evaluate the success rate of dental

implants placed after sinus augmentation with either BCP or DBB. After 8 months of graft

healing 62 dental implants with SLActive®

-surface were placed. Twenty-four implants were

placed in sites grafted with BCP, 23 in sites grafted with DBB and 15 in residual bone close to

the augmented areas. SLActive®

surfaced implants were chosen for this study since studies on

miniature pigs have demonstrated a significantly higher percentage of bone-to-implant contact

(Buser et al 2004) and better bone anchorage at an early stage of healing compared to

implants with a more hydrophobic SLA®

-surface (Ferguson et al. 2006). Moreover, clinical

studies of SLActive-implants placed in BCP and DBB are still lacking. To be able to draw

any evidence-based conclusions, the number of implants placed in BCP, DBB and residual

bone respectively should have been quadrupled. Therefore, further evidence-based studies

with a greater number of placed implants must be carried out.

After 3 years of implant loading one of the evaluation methods was resonance frequency

analysis. In the ordinary protocol the intention was to perform RFA measurement at the time

of the implant placement but it was not performed because the initial implant stability

sometimes was a risk. A mean ISQ value was calculated for each implant based on two

measurements of each implant.

Implant survival and success rates were evaluated according to criteria already mentioned.

Marginal bone loss around the dental implants should not exceed 2.0 mm after 1 year or 2.4

mm after 3 year of functional loading otherwise the implant was classified as a failure. This

may seem like a rigorous criterion for evaluation of dental implants since an implant with a

marginal bone loss exceeding 3 mm after 3 years of loading still can be functional for a long

45

period of time. Furthermore, maybe measuring marginal bone loss around dental implants is

not a valid method for evaluating success rate after only a few years of loading.

After 3 years of graft healing new biopsies were harvested for histological and

histomorphometrical analysis. Since the lateral sinus wall was intact the biopsies had to be

harvested with caution in order to avoid interaction with the previously placed dental implants.

This is of course a limitation in the possibility of harvesting representative biopsies. Due to

difficulties harvesting biopsies between implants, 8 samples contained few or no graft

particles and were not representative for paired analyses and were excluded in this study. CT-

guidance would have been preferable in order to avoid inadequate biopsy retrieval.

Comments on results

After 1 year of functional loading one implant placed in each graft was lost, giving an overall

implant survival rate of 96.8%. The success rates for implants placed in BCP and DBB were

91.7% and 95.7% respectively. Since the material tested (BCP) was new and clinical studies

were lacking, we decided to have a reasonable sample size in case of complications. Due to

the limited sample size in this study, conclusions are difficult to draw. Nevertheless, the

results for implants placed in BCP and DBB are in accordance with the general survival rate

for current implant treatment. In a study comparing implants placed in ordinary bone with

implants placed after sinus augmentation a decrease in success rate of 5-10% was shown

(Esposito et al. 2006). Results from this present study showed similar implant survival

independent of the biomaterial used. In another study in this thesis (Lindgren et al. 2009) it

was stated that bone formation mainly took place between the BCP particles and to a lesser

extent on the BCP surfaces compared to the DBB particles. This could mean that the

statistically significantly lower newly formed bone-to-BCP-particle contact does not affect the

osseous integration of the implants when used in the maxillary sinus. Moreover, the

osteoconductivity of the titanium dominated new bone formation inside the threads of the

placed microimplants in both DBB and BCP particles after 8 months of graft healing.

After 3 years of functional loading no further loss of dental implants was registered, giving an

overall implant survival rate of 96.8%. Success rates for implants placed in BCP, DBB and

residual bone were 91.7%, 95.7% and 86.7% respectively. There was a significant difference

in marginal bone level between implants placed in DBB and residual bone (p=.028). One

explanation might be that the anterior part of the maxilla had inadequate width due to atrophy

or was exposed to increased loading forces. Comparing the 1-year results (Lindgren et al

2010b) of this study with the 3-year results (Lindgren et al. 2011), no change in implant

success/survival rate was found.

In order to evaluate the sinus health CBCT was carried out. None of the patients showed any

clinical symptoms or radiological signs of sinusitis. Seventy-eight percent of the sinuses were

judged as healthy with no signs of hypertophic sinus mucosa. Four sinuses (22.2%), divided

evenly between sinuses augmented with BCP or DBB, had small mucosal thickenings within

the area of interest. This is in accordance with findings screening a normal population

(Timmenga et al. 1997).

After 3 years of functional loading the dental implants were evaluated using a resonance

frequency analyser. Similar ISQ values were found for implants placed in BCP and DBB

respectively. However, implants placed in residual bone demonstrated statistically

significantly lower ISQ-values compared to implants placed in DBB (p=.008). Since all

implants placed in residual bone were inserted in the anterior region of the maxilla this could

indicate that osseointegration might vary due to position. However, in an earlier study

(Meredith et al. 1996) no statistical difference was found between anteriorly and posteriorly

placed ITI implants (Straumann®

, Basel, Sweitzerland) after 3 months of functional loading.

46

Another possible explanation to the significantly lower ISQ values for implants placed in

residual bone might be increased loading forces.

Biopsy retrieval was carried out after 3 years of graft healing for qualitative and quantitative

analysis. Since the biopsies retrieved after 8 months of graft healing contained microimplants

the osteoconductive properties of the titanium must be considered. The titanium might have

affected the amounts of newly formed bone in the biopsies. Furthermore, the titanium threads

also occupied space within the biopsies, which poses difficulties to compare the 8-months

biopsies with the biopsies harvested after 3 years of graft healing in respect of the amounts of

newly formed bone, graft particles and fibrous connective tissue.

The BCP biopsies demonstrated a greater amount of inflammatory cells compared to the DBB

samples. This inflammatory reaction was more evident than in the 8-months biopsies. One

explanation for this could be that BCP particles are breaking up into small grains, which may

lead to a more pronounced inflammatory cell presence. The histomorphometrical results after

3 years of graft healing indicated that new bone formation around the graft particles was

similar for BCP and DBB. No significant differences could be seen within the BCP and DBB

biopsies regarding bone-to-graft contact, which is not in accordance with the findings in

earlier experimental studies (Moore et al. 1987, Daculsi et al. 1998, Jensen et al. 2009). This

could indicate that the concentration of calcium and phosphate ions during the first eight

months of healing may not be balanced for optimal bone formation (Lindgren et al. 2010a).

The initial surface of the BCP particles might be to smooth for protein adsorption and

adhesion of cells (Jensen et al. 2009). Hence, changing the surface characteristics for BCP

might increase the efficiency of the material and make it more potent. It is known that

improvement of hydrophilicity and adding microporous structure to an implant surface will

improve the peri-implant bone formation (Ferguson et al. 2006, Buser et al. 2004, Degidi et al.

2009). A similar procedure with BCP graft material could clarify if modified surface

characteristics improve the bone-to-graft contact during the initial healing process.

Paper V

Comments on aim, materials and methods

The purpose of this in vitro study was to evaluate the response of human osteoblast-like cells

(HOB) to nDP-modified and un-modified DBB and BCP scaffolds. Nanotechnology and is

attendant techniques have been used for dental implant surface modification throughout the

years. Nevertheless, attempts to apply this technique on bone substitutes are still lacking. In

the present study, nDP-modification of DBB and BCP particles was carried out through

different steps of preparation including grinding and ultrasonic technique. An initial problem

was to apply a sufficient amount of cells on the bone substitute particles to gain enough RNA

for analysis. This meant that a variety of pilot studies had to be set up before the real

experiment could start. The concentration of material used in this study might be considered

high in relation to similar studies (Mladenovic et al. 2011). Nevertheless, factors like particle

size, type of cells, culture conditions and the size of the culture plates matters for different

types of osteogenic experiments. In the present study the following osteogenic markers were

chosen for evaluation; alkaline phosphatase (ALP), ostocalcin (OC), bone morphogenetic

protein type 2 (BMP-2) and integrin alpha 10 (ITGA 10). The reasons are that during

differentiation, the osteoblasts synthesize and secrete type I collagen (Col 1), ALP and other

non-collagenous extracellular bone matrix proteins such as OC, osteopontin (OP) and bone

sialoprotein (BSP). Col 1 is expressed during the initial period of proliferation and

extracellular-matrix biosynthesis, whereas ALP is expressed during the post-proliferative

period of extracellular-matrix maturation. The expression of OC occurs during the third

47

period of extracellular matrix mineralization (Aubin 1998). Bone morphogenetic proteins

(BMP) are members of the transforming growth factor β (TGFβ) family. BMP´s have a strong

impact on mesenchymal cell to osteoblast differentiation (Deschaseaux et al 2009). ITGA 10

is an integral membrane protein known to interact with extracellular matrix (ECM) and

participate in cell adhesion as well as cell-surface signalling.

The general tendency of DBB and BCP was compared. The gene expressions between DBB

and BCP were not compared statistically due to different pore size and porosity. These

parameters can influence cell behaviour as well as gene expressions and they should be

initially controlled before comparison between the biomaterials could be carried out. This

could be considered a limitation of this study.

Comments on results

Nano-crystalline-diamond-particle-coated DBB and BCP-particles can support cell growth

and stimulate osteogenic diffentiation of HOB in vitro due to the modification of wettability.

The modification of the granula with the characterized nDP solution will certainly affect the

surface area at nano level. Previous articles have shown that a rougher scaffold area favours

osteoblast attachment and facilitates migration of osteogenetic cells and ingrowth of bone

compared to a smoother scaffold surface (Gotfredsen et al. 1992, Mustafa et al. 2000, Mustafa

et al. 2001). However, in the present study surface characterization was not carried out

initially, which makes it difficult to evaluate its importance.

In respect to HOB grown on nDP-coated and non-modified BCP particles the osteogenic

markers of ALP showed no significant difference after 1 week of incubation time. After 14

days the osteogenic markers were significantly more up-regulated in the modified group than

in the control. Nevertheless, the adhesion marker ITGA 10 was significantly down-regulated

in mRNA level for the modified group as was the the ITGA 10 level in nDP-modified DBB

after 2 weeks of incubation. For synthetic BCP the osteogenic markers for ALP, OC and

BMP-2 were all up regulated for the modified group compared with the non-modified group

after 2 weeks of incubation. The down-regulation of BMP-2 in the nDP-modified group might

be explained by the fact that DBB originates from bovine bone. The material is non-synthetic

pristine bone. The DBB-particles might already be optimized for HOB gene expression due to

a rough natural topography and wettability, which allows osteoblast attachment, proliferation

and matrix synthesis. The inner surface area of DBB is also believed to play a crucial role for

blood vessels and bone ingrowth (Weibrich et al 2000).

The results indicate that BCP is not optimized for gene expression in its present form and that

the nDP-modified BCP enhances the osteoblast phenotype and suggest that these scaffolds are

appropriate cell carriers, superior to non-modified BCP particles.

48

CONCLUSIONS

The results from the present thesis support the hypothesis that biphasic calcium phosphate can

be an alternative to deproteinized bovine bone in maxillary sinus floor augmentation

procedures to enable placement of dental implants. The results from Papers I and II emphasise

that the surface and composition of BCP-particles might not be optimized for initial bone

formation. The results from Paper V support this theory.

Paper I

A similar degree of bone formation and bone-to-implant contact was found after 8 months of

graft healing around sandblasted and acid-etched microimplants placed in sinuses augmented

with BCP or DBB. However, DBB showed a higher degree of particles in contact with newly

formed bone.

Paper II

BCP material may be chemically altered during the first 8 months of healing. The results

suggest that the β-TCP component of BCP may be gradually substituted by calcium-deficient

HA over the healing period. This process and superficial degranulation of BCP particles may

influence the progress of resorption and healing.

Paper III

After one year of functional loading no significant difference in implant success rate for

SLActive®

implants placed in augmented maxillary sinuses was found using BCP (91.7%)

compared to implants placed in DBB (94.7%).

Paper IV

A similar degree of bone formation and bone-to-graft contact for BCP and DBB was found 3

years after maxillary sinus augmentation. However, the qualitative tissue response differed.

The BCP biopsies demonstrated an extensive ongoing and greater inflammatory reaction

compared to the DBB samples possibly due to resorption of the graft particles. There was no

statistically significant difference in implant success rate for SLActive®

implants placed in

maxillary sinus augmented with BCP (91.7%) compared to implants placed in DBB (95.7%)

following 3 years of functional loading.

Paper V

Nano-crystalline-diamond-particle (nDP) coated DBB and BCP-particles can support cell

growth and stimulate osteogenic differentiation of HOB in vitro due to surface modification

and change of wettability. The results indicate that nDP-modified particles enhance the

osteoblast phenotype and suggest that these scaffolds are appropriate cell carriers, superior to

non-modified materials. The osteogenic markers of OC and ALP were increased for nDP-

modified DBB particles but BMP-2 was down regulated in relation to non-modified DBB

particles. DBB originates from pristine bone with a surface roughness and wettability that

might already be optimal for gene expression.

49

ACKNOWLEDGEMENTS I wish to express my gratitude and deep appreciation to:

My tutor, Associate Professor Mats Hallman, to whom I am deeply grateful for his skilful

guidance and support throughout this long process; your patience is enormous. Thank you for

introducing me to the scientific field and for making this possible. You are one in a million!

My co-supervisor; Professor Per Aspenberg, for his scientific support and fruitful discussions.

My co-authors:

Arne Mordenfeld, for his support with 3 of the studies and constructive ideas during this

process.

Lars Sennerby, for valuable discussions and support.

Rachel Sammons for her support and for having been the perfect host in Birmingham.

Kamal Babikeir Eln Mustafa for his support, scientific guidance and for letting me be a small

part in his pleasant department of Clinical Dentistry in Bergen.

Ying Xue, for excellent guidance in the world of bone tissue engineering.

Carina Johansson, for inspiring collaboration and for excellent help in the field of

histomorphometry.

Doris Steinmueller-Nethl, for stimulating and interesting discussions and help with the nano-

crystaline diamond-particle modification and preparation in Paper V.

Other collaborators of great importance:

Agneta Marcusson, my mentor and friend. Without you none of this would be possible.

Kristina Espemar Holst, for her support throughout paper I and for her patience with my

limited research experience.

Birgit Ljungquist for skilful support with the statistical analysis

Christopher Smith, for proofreading the frame of this thesis.

My friends and colleagues in Linköping, Gävle and Bergen

I express my sincere appreciation and thanks to my colleagues:

Sten Sahlholm, Björn Olaisson, Jahan Abtahi, Johan Nilsson, Mats Bellinetto Ericson, Reine

Sjöman, Anders Ottosson, Hartmut Feldman, Tomas Strandkvist and Anders Holmlund.

50

Torbjörn Ledin, for great statistical advice and for sharing his experiences with me.

Kerstin Schander, for inviting me to your beautiful home in Bergen and for being a true friend

and an amazing person.

I express my sincere appreciation and thanks to the following nurses:

Carina Eriksson, who helped me with the logistical work always with a smile on her face.

Lena Nordh, for assisting me throughout the clinical work and for your kindness.

Agneta Källberg, for always being one of my best friends and supportive in difficult times.

Sara Mellberg, for being the best room mate in the world.

Ann Arnell, for being who you are.

Annika Karlsson, for your excellent skills and friendly nature.

Elin Pettersson, for always being a friend and an excellent nurse.

To all my friends, you know who you are....

Stand up and be counted

For what you are about to receive

We are the dealers

We'll give you everything you need

Hail, hail to the good times

Cause rock has got the right of way

We ain't no legend, ain't no cause

We're just livin' for today

(For those about to rock: artist AC/DC, composer AC/DC)

Simon, Stefan, Fredrik, Anders, Oliver and Gaston.

Curtains drawn now it's done

Silencing all tomorrows

Forcing a goodbye

Lay down, black gives way to blue

Lay down, I'll remember you

(Black gives way to blue: artist Alice in chains, composer Alice in chains)

51

Ragne Norman, Jeanette Norman, Magnus Boman, Anna Boman and Emmy Boman

For inviting me to your family and for your kindness and support.

Eva Norman

For always being there for me during the end of this journey, I love you so much.

You say you'll give me

Eyes in a moon of blindness

A river in a time of dryness

A harbour in the tempest

But all the promises we make

From the cradle to the grave

When all I want is you

(All I want is you: artist U2, composer U2)

To my family and especially my beautiful nieces, Olivia Trabold, Indra Trabold, Liv Van

der Meulen and Josefin Van der Meulen. I´m sorry for not spending enough time with you

writing this thesis.

“Aim for the stars and maybe you´ll reach the sky”

52

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