Bone tissue regeneration in dento-alveolar surgery Clinical and experimental studies on biomaterials and bone graft substitutes Annika Sahlin-Platt

Odontological Dissertations, Series No 119 Department of Odontology Umeå University Umeå 2011

Responsible publisher under swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 12-1234-123-1 ISSN: 1234-1234-119 Cover photo: Annika Salin-Platt Elektronisk version tillgänglig på http://umu.diva-portal.org/ Tryck/Printed by: Print & media Umeå, Sweden 2011

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To Jeff, Ami, Helena and Jenny

“There are more things in heaven and earth, Horatio, Than are dreamt of in your philosophy.”

William Shakespeare

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Table of Contents Table of Contents ii

Abstract iv

List of papers 1

Abbreviations 2

Enkel sammanfattning på svenska 3

Preface 5

Introduction 6

Treatment of bone defects in dento-alveolar surgery 6

Bone tissue 8

Physiology 8

Structure and composition 8

Cells 10

Bone resorption 12

Bone formation and remodeling 12

Biomaterials 14

Dental restorative materials 14

Bone grafts substitute materials 16

Aims 18

General aim 18

Specific aims 18

Material and methods 19

Clinical treatments 19

Patient selection (Paper I) 19

Root end surgery (Paper I) 19

Radiographic evaluation of healing results (Paper I) 20

Biomaterials 20

Dental restorative materials 20

Dyract AP (Paper I, II) 20

Ketac Silver (Paper I) 21

Bone graft substitute materials 21

Bio-Oss (Paper III, IV) 21

45S5 Bioactive glass (Paper IV) 22

Algipore (Paper IV) 22

Isolation and culture of cells 23

Human periodontal ligament (PDL) cells (Paper II) 23

Bone Marrow Stromal Cells (BMC) (Paper II, III) 23

Rat Calvarial Cells (RC) (Paper III) 24

Osteoblastic mouse cell line (MC3T3-E1) (Paper III) 24

DAP procedures 24

Preparation of DAP specimens (Paper II) 24

Medium extract of DAP (Paper II) 24

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DAP direct-contact tests (Paper II) 24

Analysis of number of viable cells 25

Neutral Red Uptake Assay (Paper II) 25

Gene expression analysis 25

RNA extraction and cDNA synthesis (Paper II) 25

Quantitative real-time PCR (Paper II) 25

Bone nodule mineralization assay 25

Von Kossa staining (Paper II and III) 26

Alizarine Red staining (Paper III) 26

Elementary ion release analysis 26

Fluoride ion release (Paper II) 26

Inductively coupled plasma optical emission spectroscopy, ICP-OES (Paper II,

III, IV) 26

Medium extract of bone graft materials 26

Extract Bio-Oss, Bio active glass 45S5, Algipore (Paper III, IV) 26

Surface analysis of bone graft substitutes 27

Scanning Electron Microscopy/Energy Dispersive X-ray analysis

(SEM/EDAX)(Paper III, IV) 27

Cryogenic X-ray Photoelectron Spectroscopy fast frozen samples (Cryo-XPS)

(Paper IV) 27

Calcium 45 isotope labeling (Paper III, IV) 27

Results and discussion 28

Paper I, II 28

Paper III, IV 31

Conclusion 37

General Conclusion 37

Specific Conclusions 37

Acknowledgements 39

References 41

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Abstract Pathological processes in the alveolar and facial bones can lead to bone

loss that may not heal with complete regeneration. Biomaterials can be used to facilitate the healing process and/or as a bone substitute, but the mechanisms are not fully understood. Persistent leakage of bacteria/ bacterial toxins, after root canal treatment, may lead to a residual bone defect. The healing is dependent on a placed dental biomaterial providing a tight seal. The composition of the filling material may also influence the healing process. The general aim of this study is to investigate surface properties and biological interactions of biomaterials used in dento-alveolar surgery. A dental biomaterial, a bonded compomer (DAP) containing a corroding glass filler, was used as a root end filling material, promoting a new operation technique. The healing (assessed according to Molven´s x-ray criteria) demonstrates a significant improvement in healing results for the compomer group, compared to a commonly used technique. The surface properties and biological interactions of DAP were analyzed. ICP-OES of DAP cell culture medium extract demonstrated a significant release of Sr, Si and F from the dental biomaterial. Human periodontal ligament (PDL) cells grew on and around DAP specimens without any sign of toxic reactions. DAP extract stimulated proliferation of PDL cells, but caused an inhibition of osteoblastic gene expression in mouse bone marrow cells. The surface properties of the glass containing compomer may contribute to improved healing of the periapical lesions.

A bovine inorganic bone graft substitute (BO) is commonly used as a treatment option in dento-alveolar surgery with new bone formation in immediate close contact with BO material. ICP-OES dissolution analysis of cell culture media, after incubation with BO particles, demonstrated a dose-dependent release of Si and a decrease of Ca and P. An uptake of Ca from the medium to the BO particle was demonstrated with calcium-45 labeling. The Si dissolution varied between different batches, possibly reflecting a variation in food intake in the animals. Stimulated osteogenic response was seen in close contact to the BO particles in cell cultures. Furthermore, it was clearly demonstrated that the study design is a critical factor for correctly understanding biomaterials’ biological interactions.

The surface properties of three bone graft substitutes reported to have good results in dento-alveolar surgery were investigated, in order to establish whether or not dissolution-precipitation reactions could contribute to the bone healing. Dissolution-precipitation extracts of BO, bioactive glass 45S5 (BG) and a marine algae hydroxyl apatite (AP) in cell culture media were analyzed. Dissolution of Si at significant levels was detected for BO and 45S5

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over time. Significant uptake levels of Ca and P from the culture were seen for both 45S5, BO and AP but at different times. Surface analysis of the biomaterials with SEM/EDAX, before and after immersion in cell culture media, revealed a smoothing of the surface morphology for 45S5 over time. No obvious alterations for BO and AP were detected. Ca/P ratio decreased significantly for 45S5, but no major changes were detected by XPS for BO or AP. XPS further demonstrated a surface charge for BO, changing from negatively to positively charged when exposed to serum. 45S5 and AP had positive surface charges, both in the absence and the presence of serum. These demonstrated surface changes in biomaterials could contribute to adherence of cells and subsequently affect bone healing.

Conclusion: Biomaterials used in dento-alveolar surgery interact with biological surroundings through surface and dissolution-precipitation reactions which may have implications for bone healing.

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List of papers

This thesis is based on the following original papers, which will be referred to by their Roman numerals:

I. Sahlin-Platt A, Wannfors K. The effectiveness of compomer as root-

end filling: A clinical investigation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:508-12.

II. Sahlin-Platt A, Örtengren U, Mladenovic Z, Ransjö M. Effects of Dyract AP and released ionic products on periodontal ligament cells and bone marrow cultures. Dental Materials 2008;24:1623-1630.

III. Živko Mladenović, Annika Sahlin-Platt, Britta Andersson, Anders

Johansson, Erik Björn, Maria Ransjö. In vitro study of the biological interface of Bio-Oss®: Implications of the experimental setup.

Clinical Oral Implant Research. Accepted

IV. Mladenović Ž, Sahlin-Platt A, Andersson B, Shchukarev A, Björn E, Bengtsson Å, Ransjö M. Ion exchange and surface reactions in three bone graft substitute materials after incubation in cell culture medium. In manuscript. Submitted

The original papers are reprinted with the kind permission from the publishers.

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Abbreviations 1,25(OH)2D3 1,25-Dihydroxycholecalciferol ALP Alkaline phosphatase α-MEM α-modification of Minimal Essential Medium BMP Bone Morphogenetic Protein

BSP Bone Sialoprotein β-TCP β-tricalcium phosphate BMC Bone Marrow Cells Cbfa-1 Core binding factor α-1

CSF Colony Stimulating Factor DFDB Demineralized Freeze Dried Bone, DMEM Dulbecco’s modified eagle’s medium FBS Fetal Bovine Serum FCS Fetal Calf Serum FHA Fluoro hydroxyapatite HA Hydroxy apatite LED Light-Emitting Diode

MCSF Macrophage Colony Stimulating Factor MMP Matrix Metalloproteinase MSC Mesenchymal Stem Cell OPG Osteoprotegrin

OPN Osteopontin PAI Periapical index PDGF Platelet-derived growth factor PTH Parathyroid hormone RANK Receptor activator of NFκB RANKL Receptor activator of NFκB ligand RGD Arginine-glycine-apartic acid RUNX 2 Runt-related transcription factor 2 QHT Quartz-Halogen-Tungsten light curing unit SBF Simulated body fluid

TCB Tetracarboxylic acid-hydroxyethyl methacrylate-ester TEGDMA Triethylene glycol dimethacrylate TGFβ Transforming growth factor β TNAP Non tissue specific alkaline phosphatase

TRAP Tartrate resistant acid phosphatase UDMA Urethane dimethacrylate UHV Ultrahigh vacuum VEGF Vascular endothelial growth factor WNT Wingless-int XPS X-ray Photoelectron Spectroscope

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Enkel sammanfattning på svenska

Det övergripande syftet med denna studie är att undersöka biomaterialens egenskaper och de biologiska utbytena med vävnad som ligger i omedelbar kontakt med materialen vid käkkirurgi.

Patologiska processer i käkbenet och andra ansiktsben kan leda till benförlust som inte läker helt utan att viss benförlust blir permanent. Man kan då använda biomaterial som ett substitut för benförlusten men hur mekanism för hur biomaterialen fungerar är inte helt klarlagt. Kvarstående bendefekter efter rotbehandling kan bero på läckage av bakterier och/eller deras toxiner från rotkanaler. Ett rotspets material kan genom en operation läggas över rotspetsen på tanden. Läkningen är dels beroende av om materialet tätar rotkanalen optimalt och om materialets beståndsdelar påverkar läkningsprocessen.

Ett biomaterial för tänder, Dyract AP (DAP), som innehåller fillerpartiklar av korroderande kisel-fluor-strontium glas användes till att försluta rotfyllningen vid rotspetsen tillsammans med en anpassad operationsteknik. Läkningsresultaten (enligt Molvens röntgenkriterier) visade en signifikant förbättring av benläkningen i den patient grupp som behandlats med DAP jämfört med rutinmässig operationsteknik. Ytegenskaper och biologiska interaktioner hos DAP analyserades i en experimentell studie. Med ICP-OES analys av extrakt från DAP provkroppar i cellodlingsmedium kunde man visa signifikanta men relativt små mängder strontium, kisel och fluor frisatt från DAP. Humana rothinneceller (PDL) växte på och runt DAP material utan tecken på reaktion från något giftigt. Extrakt från DAP stimulerade tillväxt av PDL-celler men hade en hämmande effekt i en experimentell benbildnings modell benmärgsceller hos mus. Den goda läkningseffekten på patientmaterial kan antingen bero på att DAPs täta förslutning mot tandytan eller ytegenskaperna hos DAP materialet.

Ett bovint oorganiskt benersättningssubstitut Bio-Oss (BO) är vanlig biomaterial/benersättningsmaterial vid käkkirurgisk behandling av bendefekter. Nybildat ben i omedelbar anslutning till biomaterialet har tidigare påvisats med benbiopsier vilket antyder att materialegenskaper kan påverka benläkning. ICP-OES jon analys av medium efter inkubation med BO partiklar påvisade en dosberoende frisättningen av kisel som varierade med olika batcher. Upptag av kalcium och fosfor från mediet till BO partiklarna kunde påvisas, detta kan ha betydelse för benläkningsprocessen. Stimulerande effekt av BO kunde ses i en experimentell benbildningsmodell.

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En väsentlig slutsats från studien är att biomaterial påverkar omgivningen i vävnaden och att designen av experimentella studier måste inkludera detta faktum.För att ytterligare förstå hur biomaterial kan fungera som benersättningsmaterial vid regenerativa operationer i käkarna undersöktes ytegenskaperna hos tre inom odontologin använda benersättningsmaterial (Bioaktivt glas 45S5, Bio-Oss samt Algipore). Materialen har olika ursprung men visar alla god ben nybildning i omedelbar anslutning till materialet. Jonfrisättnings analyser visade ett upptag av kalcium och fosfor på ytan av Bio-Oss och Algipore samt upptag av fosfor och frisättning av kisel från det bioaktiva glaset 45S5. Med ytanalyser på mikro till nano nivåer fastställdes att biomaterialens ytor var laddade och att proteiner band in till ytan när materialen inkuberades i medier med serumproteiner.

Konklusion: Biomaterial som används inom käkkirurgi interagerar med omgivande vävnad genom ytreaktioner beroende på biologiska förutsättningar, vilka kan ha betydelse för benläkning.

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Preface This has been a journey, not only from Stockholm to Umeå, but also into

the fascinating and sometimes frustrating world of research. However, it has also been a journey from the very tangible world in the clinical work with patients, to the micro cosmos of the first few atomic layers in the interface where biomaterial meets biology. It all belongs together and the micro cosmos is most certainly a very real part of our daily work as clinicians. When I was working at the maxillofacial department at Södersjukhuset, Stockholm I was performing dento-alveolar surgery. One of the treatments was retrograde root resections with an application of a root end filling material. I was intrigued that one of the techniques of choice involved using a moisture sensitive root end filling material. The contamination of the filling material by the fluids in the operation wound could possibly mean an impaired healing result due to material defect. I felt that an alternative filling material that might be better controlled during the procedure was needed. This was the start of an investigation of materials with quite different material properties. Furthermore, this also meant a need for an alternative operation technique. The results of a primary study of an alternative dental filling material raised the question whether the composition of the filling material in itself contributed to the bone healing. This would mean that the material could promote bone healing in a more active way by influencing the surrounding tissue possibly from products leaching from the material. This study on root-end resections led to the start of the studies presented in this thesis, on different materials used in dentoalveolar surgery. The effects of the material’s composition and properties on cells of these materials are studied. Since the time from the start of these studies, the notion of tissue engineering using a new form of materials, biomaterials, has developed as an important part of reconstructive surgery of bone defects.

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Introduction

Treatment of bone defects in dento-alveolar surgery Bone has a remarkable healing potential (Lemperle el al., 1998). The newly formed bone integrates sufficiently with the same mechanical and biological properties after maturation, as the surrounding bone tissue (Marsell and Einhorn, 2011). Impaired blood clot formation may cause reduced bone formation during healing (Cardaropoli et al., 2003) and also after infections such as osteomyelitis, apical residual cyst, periodontitis (Baldini et al., 2011)and apical periodontitis. The bone defect, depending on the size and form, may be necessary to supplement with regenerative methods (Kneser and Stark, 2002). In these cases a surgical intervention can be needed to correct the lack of new bone. Bone in the alveolar ridge undergoes natural resorbtion as teeth are lost (Araujo and Lindhe, 2005, Soucacos et al., 2008). In cases of a heavily resorbed alveolar ridge, a bone graft or a bone graft substitute biomaterial can be used to reconstruct bone tissue (Esposito et al., 2006).

Fig.1. Bone loss due to; a) osteomyelitis, b) periapical cyst c) periodontitis

d) apical periodontitis. (X-ray used with permission from the patients)

Bone grafting refers to a surgical procedure, where bone from a donor site is

transplanted to a recipient site (Stedman, 2000). Different grafting methods have been used in bone surgery for regenerative purposes after completed infection control. The vascularisation of the graft and the process of hematoma formation are essential for new bone formation. Graft material of human origin is either from the patient´s own skeleton (autogenous bone) or donated from another human

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(allograft). Replacing lost bony tissue with a bone graft from the patient’s own bone has been considered the golden standard (Soucacos et al., 2008). The autogenous bone graft has the possibility for surviving bone cells in the graft, to differentiate into cells with bone producing potential in the recipient site. The autogenous bone graft has the advantage of causing limited immunological reactions as the graft is harvested from the patient´s own bone in the donor site. Another advantage is the presence of growth factors in the graft that might enhance new bone formation in the recipient site. Depending on the amount of bone graft needed an additional operation site might be required. Adverse reaction to the autogenous bone graft is limited, but the additional operation site for harvesting the graft might cause the patient increased post-operative discomfort and a risk of donor site morbidity. An extensive resorption of the autogenous bone graft may result in an unpredictable volume of newly formed bone (Dimitriou et al., 2011). In addition, the increased cost for a donor site operation in terms of time and money is an aspect that could influence the choice of surgical method (Dahlin and Johansson, 2010). An alternative to autogenous bone graft is using bone from a donor, an allograft. The allograft can either be transplanted without removing any parts of the bone tissue (fresh or freeze dried) or it can be processed to remove any minerals leaving proteins, growth factors and the bone matrix (demineralised freeze dried bone, DFDB). Concerns have arisen regarding allograft, with respect to the possible contamination by viruses from donor material to the host, thus promoting the development of synthetic bone graft substitutes as an alternative (Brant and Davison, 2008). Another option is using a xenograft, a graft material from another species, either animal or plant. The use of synthetic biomaterials, for promoting regeneration of bone tissue, is gaining ground as an accepted valid alternative method, in a wide range of surgical procedures.

The object of using biomaterials in bone surgery is to enhance the healing potential of bone defects without the additional surgical intervention of harvesting a bone graft from the patient. Definition of biomaterial was described by D.F. Williams 1999: ‘‘Materials intended to interface with biological systems to evaluate, treat, augment, or replace any tissue, organ or function of the body.’’(Williams, 2009). The definition of a biomaterial has changed over time as the development of the science of tissue engineering and the understanding of interactions between bone biology and biomaterials has progressed. A more recent definition of biomaterial is an implantable material that performs their function in contact with living tissue (M. Vallet-Regí, 2010). The most common graft materials used in oral surgery are hydroxyapatite or a synthetic fast resorbing β-tri calcium phosphate. Naturally derived biomaterials containing hydroxyapatite (such as deproteinized bovine bone) or calcium carbonate (as in marine algae, Coralina officinalis) are commercially available and used in oral surgery. Successful bone healing depends on the restoration of biological processes, morphological structure and mechanical function in the newly formed bone. To

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further understand these processes, research is required regarding the interactions at the interface between bone and the biomaterial. Furthermore, new materials with specialised functions targeting specific treatments needs might be possible to develop with increased knowledge regarding composition and function of the biomaterials. It is therefore important to understand the underlying biological- chemical interactions in the biomaterials used today.

Bone tissue

Physiology

Bone is an important source of calcium and a part of the regulatory system for calcium homeostasis. Uptake of calcium ions (Ca) from the intestines, kidneys and release from the skeleton is regulated by systemic hormones. Calcium ions are vital for many cell functions and play an important role in signal transduction pathways. Ionised calcium is present in blood plasma as well as in the tissue fluid outside cells and in the cell cytoplasm. The blood concentration of Ca is highly controlled by calcium regulating hormones; parathyroid hormone (PTH), vitamin D and calcitonin. A low concentration of Ca in blood activates PTH secretion from the parathyroid glands. A high concentration of Ca in blood activates calcitonin secretion by the thyroid gland. The absorption of Ca from the intestines and calcium release from the skeleton is regulated by Vitamin D3 and is a well defined feedback homeostatic system that sets the Ca concentrations in blood to 2.2-2.5 mMol/l. Phosphorous is the most abundant ion in all tissues and has important functions in providing high energy in cell metabolism, influencing enzymatic reaction and protein functions. Phosphorous is also essential component in cell membranes and in the mineral phase of bone, e.g. hydroxyapatite.

Structure and composition

The skeletal structure is designed, through its different bones, as a physical support to muscles and tendons in the locomotors system and as a physical protection for the inner organs. The skeletal bones also harbour bone marrow which is a supplier of blood and bone cells (Fig 2).

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Fig. 2. Schematic figure showing the structural composition of bone.

Figures were produced using Servier Medical Art

Bone is, by its structural composition, divided in to trabecular and compact bone. Compact bone, also referred to as cortical bone, can be seen in the long bones and the surface of the flat bones. Trabecular bone is a lacework surrounding bone marrow within most flat bones and the metaphyseal region of the long bones. Both trabecular and compact bone have the same composition of cells and matrix, but differ in structural design and function. The newly formed bone is reorganized through remodeling processes forming a cone shaped structure along a blood vessel, the Haversian canal. The mature bone has a laminar appearance with enclosed bone cells. Microscopic canals form connections between the cells and from the cells to the bone surface (Buckwalter 1995) (Fig 2).

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Bone is made up of a mineralized fibrous matrix including bone cells and water. The inorganic part of bone (70%) is mainly calcium and phosphate, the Ca/P ratio being 1.67. The minerals form a crystalline structure of hydroxyapatite [Ca10(PO4)6(OH)2]. Additional minerals e.g. silicon, strontium and fluoride are absorbed from the bloodstream and incorporated in the bone mineral phase. It is suggested that these elements can affect the bone cell metabolism altering the osteoblast /osteoclast function (Gazzano et al., 2010). The organic part of bone is an extracellular matrix (ECM) consisting of several proteins. The ECM is essential for the mechanical properties, the metabolic function and the formation of bone. The main part of the ECM is collagen type 1 (>90%) The formation of collagen fibers in the bone matrix is essential for bone formation. There are more than twenty different types of collagen with a variety of biophysical and biochemical properties such as solubility, low immunogenic properties, and high tensile strength. Collagens form structural support to the skeleton, skin, blood vessels, nerves and fibrous capsules of organs (Patino et al., 2002, Allori et al., 2008b). The remaining part bone EMC, apart from collagens, are non-collagenous proteins such as proteoglycans (decorin, biglycan, osteoadherin) and glycoproteins (osteonectin, osteopontin and bone sialoproteins). Osteonectin binds to collagen type I and regulates mineralization with a high affiliation to Ca in hydroxyapatite (Hoang et al., 2003) In the ECM a multitude of growth factor groups and their isoforms act as biological signals in bone formation. Some of these growth factors - vascular endothelial cell growth factor (VEGF), transforming growth factor β (TGF-β), platelet derived growth factor (PDGF) and the bone morphogenetic protein family (BMP) are closely involved in bone formation and bone turnover and could have implications for regenerative surgery (Allori et al., 2008a).

Cells

The main bone cells are osteoblast, osteocytes and osteoclasts. Osteoblasts derive from mesenchymal stem cells (MSC), which can be found as bone marrow (Askmyr et al., 2009). MSC can, apart from osteoblasts, differentiate into adipocytes, fibroblasts, chondrocytes and myoblasts. The determination of the final cell type is dependent on factors regulating intracellular signaling pathways and finally the gene expression specific for each cell. One important regulator of osteoblast differentiation is the osteoblast specific gene transcription factor Runx2/Cbfa-1. Osteocytes, the most common cell in the bone tissue, are osteoblasts embedded in the bone matrix in lacunae surrounded by mineralized matrix (Fig. 3). Osteocytes have protoplasmic extensions reaching through the microscopic canaliculi (Bonewald and Johnson, 2008). The extensions are in contact with osteoblasts on the bone surface or other osteocytes through gap junctions, a cell-cell communication channel (Civitelli, 2008). This ostecyte cell-cell communication is considered to be essential for the bone response to loading forces (Skerry, 2008). Lining cells are flat inactive osteoblasts covering all bone surfaces. Osteoblast and bone marrow stromal cells express factors initiating signaling programs which are

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crucial in the formation and activation of bone resorbing osteoclasts (Matsuo and Irie, 2008).

Fig 3. Osteoblast/osteoclasts recruitment, activation and interactions.

Figures were produced using Servier Medical Art

Osteoclasts are highly specialized multinucleated bone resorbing cells originating from the monocyte-macrophage cell line. The osteoblasts and bone marrow stromal cells stimulate the differentiation and fusion of pre-osteoclast to multinucleated osteoclast by expressing macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL), two cytokines essential for osteoclastogenesis. The interaction between RANKL and the receptor RANK expressed on pre-osteoclasts and osteoclasts activates intracellular signaling pathways and gene transcription factors important for osteoclast formation and activation. Osteoblast/stromal cells also express a decoy ligand, osteoprotegrin (OPG), which inhibits osteoclast formation/activation by blocking RANKL from binding to RANK (Fig 3). The RANK/RANKL/OPG system is an important regulator of both physiological and pathological bone resorbtion and the skeletal remodeling (Vaananen and Laitala-Leinonen, 2008).

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Bone resorption Before the osteoclasts can attach to the bone surface, the unmineralized protein layer (osteoid) covering the surface has to be degraded by proteolytic enzymes. Osteoclasts use a specific cell membrane sealing zone, in order to attach to the exposed mineralized bone surface, connecting to proteins with the arginine-glycine-apartic amino acid (RDG) sequence. This seal facilitates the development of a closed micro environment for the degradation of bone matrix and bone mineral. After attachment, the osteoclast develops another highly specialized region of the cell membrane, the ruffled border, which is a folded cell membrane essential for the resorption mechanism. The mineralized compound of bone is dissolved by a low pH created by protons secreted from the ruffled border into the space enclosed by the sealing zone (Fig.3). The organic part of the bone tissue is removed by various enzymes secreted from osteoblasts (collagenase) and osteoclasts (Cathepsin K, TRAP) (Vaananen and Laitala-Leinonen, 2008, Cohen, 2006). Bone formation and remodeling Bone can develop through two different mechanisms; endochondral bone formation starting with a template of cartilage or intramembranous bone formation whereby the bone is formed directly. The long bones in the skeleton are formed through endochondral bone formation, whereas the maxilla, the mandible and the calvarium are examples of bone formed through intramembranous ossification. In endochondral ossification, the cartilage matrix mineralizes in the centre initiated by vascular ingrowth. An invasion of osteoblastic precursor cells differentiates into osteoblasts and forms bone, which replaces the cartilage to the full, except for the epiphyseal growth plate (Cohen, 2006, Kronenberg, 2003)(Fig. 4).

Fig 4. Endochondral bone

formation.

Figures were produced using

Servier Medical Art

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Intramembranous bone formation starts with a condensation of osteoblastic precursor cells. When the cell-condensation reaches a high enough density the differentiation into an osteoblastic lineage starts (Fig. 5).

Fig. 5. Intramembraneous bone formation. Figures were produced using Servier Medical Art

Osteoblasts lay down an extracellular matrix mainly consisting of collagen Typ 1, but also non collagenous proteins, important for the mineralization process. During bone formation, osteoblasts become embedded in the mineralized tissue and become osteocytes. The bone surface is covered by an unmineralized protein layer, osteoid (Franz-Odendaal, 2011). The skeleton is renewed in a continuous process as a part of repair and structural increase, in a response to loading. This remodeling exchanges about a tenth of the bone volume per year. A balanced bone remodeling is dependent on the recruitment of precursor cells for both osteoclasts and osteoblast and coordination between the activities of the two cell types. Direct or indirect signals between osteoblast-osteoclast ensure the balance in the bone turnover (Fig. 3, 4). Reversal cells, of unclear origin, are suggested to prepare the bone surface for osteoblast bone formation (Fig. 6). The activation of the osteoblasts/bone marrow stromal cells is suggested to involve freed molecules from the newly degraded bone tissue or possibly direct from osteoclasts (Raggatt and Partridge, 2010). An imbalance in this system can cause pathological changes to the bone as seen in osteoporosis, osteopetrosis and Paget’s disease.

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Fig.6. Bone remodeling. Figures were produced using Servier Medical Art

The mechanisms of the mineralization process of bone are not fully understood. The pre-mineralized matrix contains, apart from collagens, proteoglycans and acidic non-collagenous proteins. An under group of the acidic non-collagenous proteins are SIBLINGS (Small Integrin-Binding LIgan, N-linked Glycoprotein) such as Bone sialoprotein (BSP) and Osteopontin (OPN) connected with bone mineralization by possible Ca2+ attracted to the acidic proteins. BSP is suggested to promote mineralization and OPN both promoting and inhibiting the mineralization process. It has also been suggested that the mineralization starts as calcium /phosphate mineral deposition in matrix vesicles “budded” from the plasma membrane of osteoblasts and deposed in the empty void in between the collagen fibers . Nano crystals grow out of the vesicles and hydroxyapatite is formed in the extracellular matrix (Golub, 2009, Simao et al., 2010). The protein, alkaline phosphatase (ALP), is promoting mineralization through increasing phosphate levels by the non tissue specific isoform, TNAP. TNAP is expressed by osteoblast progenitor cells, osteoblasts and young osteocytes. TNAP also inactivates the mineralization inhibitor pyrophosphate. ALP is a late marker for osteoblast activity in cell cultures (Allori et al., 2008a)

Biomaterials

Dental restorative materials

Loss of tooth substance (due to caries or trauma) may enable penetration of bacteria into the pulp causing an infection The bacteria or bacterial toxins may leak

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into adjacent periodontal tissues through the apical foramina , accessorial canals or exposed open dentine tubules. The leakage of bacteria/toxins may start an inflammatory response in the periapical area resulting in the degradation of the periodontal ligament and adjacent bone, i.e. a periapical lesion. The treatment of choice is the removal of the infected pulp tissue in the root. In case of failure, the continuous bacterial or toxins leakage increases the periapical degradation process. A surgical intervention, when orthograde retreatment of the root canal is technically or impractically difficult, could therefore be an alternative (European Society of Endodontology, 2006)

Dental materials are used in reconstruction after loss of tooth substance but can also be used in dental surgery as root end fillings. Infected root canals are mostly treated with a placement of a root filling after the root canal has been disinfected. In some cases infection persists and may be inaccessible and a surgical procedure is necessary. A dental filling material, i.e. a dental biomaterial is placed, in a prepared cavity in the apex of the dental root, as a seal to prevent leakage of bacteria or their toxins from the apical area. The only purpose of the dental biomaterial has been to act as a barrier to prevent leakage from the root canal to the surrounding periodontal tissues and thereby facilitating healing of any persistent bone defect. The use of an operation microscope is regarded as an important aid for the inspection of and the locating of all possible sites for bacterial leakage on the denuded root surface. Root end filling materials that have been used are Ketac Silver, a metal reinforced glass ionomer (no longer manufactured), Super EBA, a polymer reinforced zinc oxide, IRM (intermediate restorative material) and amalgam. Mineral Trioxide Aggregate (MTA), a tricalcium silicate, tricalcium aluminate and tricalcium oxide is now a widely used root end filling material (Kim and Kratchman, 2006, Ma et al., 2011). MTA has been reported to have high biocompatibility and promote periapical bone healing. Retroplast, a chemically cured two-component, BisGMA/TEGDMA, composite resin with the dentine-bonding agent, Gluma has also been specially developed to be used as a root end filling material (Yazdi et al., 2007). Dyract AP, which is a hybrid material consisting of a combination of composite and glass ionomer, has been suggested in experimental studies to be an alternative root end filling material (Greer et al., 2001). Dental composites, such as Retroplast, consist of a matrix with combinations of aromatic or aliphatic di-acrylates resin and inert filler particles of glass coated in silane. The resin consists of different oligomers forming chains and polymers and monomers which cross-link between the chains. Polymers and monomers also link to the glass filler via a silane layer added to the surface of the particles (Zimmerli et al., 2010, Soderholm et al., 1984). The composite resins are either light cured or chemically cured, with the degree of curing determining the remaining amount of residue monomers (di-acrylates) in the composite (Fig.7a). Monomers such as TEGDMA, UDMA and HEMA are known to be toxic and are easily dissolved in fluids (Ortengren et al., 2001, Michelsen et al., 2003). The toxicity of the composites can be tested by studying the effect of these eluates in cell cultures (ISO 7405).

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(a) (b)

Fig.7 Schematic illustration of light cure polymerisation of (a) composite, (b) acid-

base reaction of glass ionomer.

Bone grafts substitute materials

In the beginning, the desired property of the bone graft substitutes was to replace lost bone tissue with little tissue response e.g. an inert material. The development from these materials furthers the properties of a bond between the bone graft substitutes to bone through an interface. The biomaterial may act as a scaffold/physical support (osteoconductive) for the bone structure. A biomaterial may have an osteogenic capacity (osteoinductive) if the biomaterial can stimulate osteoblast differentiation and new bone formation (Van der Stok et al., 2011). The induction of new bone formation is dependent on several properties of the biomaterial such as surface topography, dissolution and precipitation reactions (Hoppe et al., 2011, Wennerberg and Albrektsson, 2009). The development of a calcium phosphate rich layer on the surface of the bone graft substitute is suggested to be essential for the formation of the interface and bond to bone (Barrere et al., 2006). With regards to biomaterial used in bone surgery, the concept of bioactivity has further developed to include the forming of a bone–like apatite layer on the surface of the material when implanted in the body (Kokubo and Takadama, 2006). Synthetic bone graft materials can be manufactured from a variety of biocompatible materials as bio-degradable polymers, bio ceramics and bioactive glasses. The materials can be produced in a variety of shapes and forms. The scaffolds can differ in degradability and thereby vary from long lasting to short term. The structure of the graft may have macro–meso porosities, which facilitate the ingrowth of blood vessels and cells (Wahl and Czernuszka, 2006). The biomaterial’s chemical composition, topography and electrical charges together with

17

the components and pH of the biological environments will determine dissolution and precipitation reactions and the biological response (Nel et al., 2009, Meyer et al., 2005). The interface between a bone graft substitute material and the surrounding tissues is a key factor for adhesion and differentiation of cells and subsequent bone formation. The study of a biomaterial’s surface could therefore give a deeper knowledge of the mechanism of the interface and the effect on bone formation. Examples of clinical use of the bone graft substitutes Bioactive glass (45S5)(Fig.8),Bio-Oss (Fig.9) and Algipore (Fig.10). Fig. 8 Example of Bioactive glass (45S5) used as bone graft substitute for bone

regeneration in periodontal surgery. At base line (a), post operatively at 1 year

(b), 2 years (c) and 3 years (d). (X-ray used with permission from the patients)

Fig.9 Example of Bio-Oss

used as bone graft

substitute for bone

regeneration for alveolar

ridge augmentation. At

base line (a), post

operatively at 1 year (b)

and 2 years (c). (X-ray

used with permission

from the patients)

Fig.10 Example of Algipore used as

bone graft substitute for bone

regeneration. At base line (a), 1 year

(b). (X-ray courtesy of Dr Ann-Marie

Roos-Jansåker)

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Aims

General aim

The general aim of this study is to investigate properties of biomaterials used in dento-alveolar surgery that may promote bone healing and bone regeneration.

Specific aims

� To investigate if a dental compomer biomaterial with an adhesive combined with an adjusted operation technique would increase healing results in root end surgery as compared to a conventional root end operation with a glass ionomer material.

� To investigate possible dissolution of inorganic ionic products from the glass component in the dental compomer incubated in a biological medium, and possible effects on cell proliferation/differentiation and bone mineralization in vitro.

� To investigate surface reactions in a deproteinized bovine biomaterial (Bio-Oss), incubated in a biological medium, and the possible effects on bone mineralization in vitro.

� To investigate surface reactions in three different biomaterials; deproteinized bovine (Bio-Oss), Bioactive glass (45S5) and porous algae-derived hydroxyl apatite (Algipore), incubated in a biological medium.

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Material and methods

Clinical treatments Root end operations were performed to remove persistent apical infections after failed root canal treatments in inaccessible root canals. Root end filling materials (Dyract AP with Prime&Bond NT and Ketac Silver) were applied in a apical cavity according to each material’s operation protocol. Radiological and clinical evaluation was performed one year after operation to assess the healing result.

Patient selection (Paper I)

Patients were referred to the Department of Maxillofacial Surgery for root end resection due to persistent periapical lesion. 34 single rooted teeth, in 28 patients, with an age range of 33-83 years, considered inaccessible for orthograde root canal treatment were randomised in the study. The patients were in good health according to self reported declaration. No inflammatory diseases or medication impairing the healing results after surgery. Computer generated randomisation of the patient material was performed.

Root end surgery (Paper I)

A full thickness flap operation was performed, buccal bone was removed and the apex of the root was exposed and resected. The commonly recommended surgical protocol for root end operation is a 3 mm root end resection, a longitudinal cavity preparation in the root canal, and placement of a root end filling material. In Paper I, a modified operation technique was used to enable the use of a light cured compomer, Dyract AP (DAP) and the bonding system Prime&Bond NT (P&B NT). DAP and P&B NT were placed in a shallow saucer shaped root end cavity (Fig 11). DAP was light cured for 60 seconds with Spectrum Curing Light System and polished with a Werhaug #2 diamond.

Fig.11 Schematic illustration to root end

preparation technique.

Root filled tooth with gold post (left),

saucer shaped (middle) and cylinder

(right) root end filling

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Ketac Silver was used as a retrograde filler material in preparation according to the common technique (control group). The material was allowed to set for seven minutes before the flap was sutured. Healing results for DAP and Ketac Silver were evaluated with clinical and x-ray examination after 1 year.

Radiographic evaluation of healing results (Paper I)

Standardised x-ray examination was performed using a Eggen-holder was performed at the time of surgery and one year post operatively. The x-rays were evaluated according to Molven´s modified periapical index scores (PAI index) by an independent oral radiologist (Orstavik, 1988).

Biomaterials

Dental restorative materials

The dental materials Dyract AP (DAP) and Ketac Silver were used in the clinical study as a root end filling material (Paper I). DAP specimens were used for evaluating cell viability and growth pattern in contact with the material. DAP extract was used for analyzing effects of dissolution products on human periodontal ligament (PDL) cells or mouse bone marrow cells (MBC). The effects of DAP extracts on mineralization and cell growth were analysed with the bone nodule and Neutral Red assays. Inorganic ions were analysed in the DAP extract with ICP-OES and ASTM methods (Paper II).

Dyract AP (Paper I, II)

Dyract AP (DAP, Dentsply/DeTrey) is a urethane dimethacrylate (UDMA) and tetracarboxylic acid-hydroxyethyl methacrylate-ester (TCB) based compomer combining the properties of a glass ionomer with that of a composite. The material contains reactive glass particles of strontium-aluminum-fluoro-silicate glass. The light induced polymerisation of monomers precedes the delayed acid base reaction as polyacrylic acid and the glass material reacts with the surrounding water. Fluoride is released from the glass particles and the material expands as water is absorbed. A photo initiator starts the light polymerisation (Fig.12). DAP, in conjunction with the adhesive system Prime&Bond NT (Dentsply/DeTrey) was used as a root end filling material. Prime&Bond NT is light cured dental adhesive with a liquid part containing cetylamine hydrofluoride and a solid component of amorphous silicon dioxide nano fillers. The DAP with colour B1 was used in the root end filling Paper I. For the DAP specimens in Paper II colour A3 was used.

21

Fig.12 The initial light curing and the later acid–base reaction of Dyract AP

Ketac Silver (Paper I)

Ketac Silver Aplicap (3M/ESPE Dental AG) is silver reinforced glass-ionomer cement earlier often used as a dental biomaterial sealing the tooth apex when apicoectomy was performed. The material consists of an aqueous part of polyacrylic acid and a solid part (the powder) of calcium fluoro-aluminum-silicate glass fused with silver (40 % by weight) (Sarkar, 1999). The acid reacts to the basic calcium in the glass, cross-linking to form a matrix. Glass ionomer cement has the ability of chemical bonding to calcium hydroxyapatite in teeth (McKinney et al., 1987). Chemical cured glass ionomer materials are not, however, water stable until 6 hours after setting and the matrix structure and the adhesion can therefore be negatively influenced by early contact with water (Nicholson and Czarnecka, 2004, Sarkar, 1999).

Bone graft substitute materials

Bio-Oss (Paper III, IV)

Bio-Oss® (Geistlich Pharma AG) is a spongeous bone substitute derived from deproteinised bovine bone (Benke et al., 2001) as granulates 0.25 -1 mm. Through chemical and heating processes the proteins and other organic substances are

removed, leaving a scaffold of interconnected hydroxyapatite. The lattice structure is similar to the trabecular architecture of human bone with a high porosity and a pore size of 300-1500µm.

Fig.13 Biopsy from alveolar ridge augmentation

with Bio-Oss after 12 months. Newly formed

bone seen in close contact Bio-Oss particles. Htx-

Eosin staining. Original magnification x 40

22

Bio-Oss is widely used as a bone graft substitute in dento-alveolar surgery (Baldini et al., 2011). Histological studies have shown formation of new bone in close contact to the Bio-Oss particles (Hallman et al., 2001, Mordenfeld et al., 2010, Iezzi et al., 2007, Iezzi et al., 2008). The close relation between the bone graft substitute material and the newly formed bone raises the question whether or not Bio-Oss may have possible osteogenic properties (Fig. 13).

45S5 Bioactive glass (Paper IV)

Bioactive glass (45S5) is a synthetic bone graft substitute with the composition 45% SiO2, 24.5% Na2O, 24.5% CaO, and 6% P2O5 . 45S5 is processed by melting the substances at 1400 ̊C to a glass mass. The glass is ground to a particles size of 70-700µm. In contact with a fluid, a corrosion process takes place, releasing elementary ions and degrading the bulk of the biomaterial (Hench, 1998, Jones et al., 2001).

Fig.14. Light microphotograph of a

histological biopsy from a patient at 9

months after surgery. Osteoid and newly

formed bone is seen inside the corroded

Bioactive glass 45S5 particles. Htx-Eosine

staining. Original magnification x 20

Bioactive glass 45S5 was developed by Larry Hench in the early 70’s. The material degrades by corrosion, a chemical and

physical surface reaction, silica glass compound absorbs from the bulk into the surrounding tissue leaving a calcium- phosphate rich layer on the surface.(Hench, 2006). Osteoblasts lay down an osteoid on the calcium phosphate surface, a seamless transition from the biomaterial to the bone is formed (Fig14). Bioactive glass 45S5 granulates corrode at a speed of 150µm in three months as shown in a dog model (Schepers et al., 1998) . The stability of the glass crystal is determined by the amount of silicon in the glass. Higher concentration of silicon results in a more stable crystalline structure.

Algipore (Paper IV)

Frios®Algipore®, (DENTSPLY, Friadent) granulates (0.5-1 mm) is a highly porous biomaterial, the porosity means diameter 10 µm. Algipore is derived from calcifying marine algae, Corallina officinalis (fig.15). Through a heating process, the algae is segmented in to calcium carbonate and after further refinement processes, part of

23

the calcium carbonate transforming into fluoro hydroxyapatite (Schopper et al., 2003).

Fig.15 Light microphotograph of a

histological section from a rat treated with

Algipore. Newly formed bone in close contact

with Algipore particles. Original

magnification x 20. (Image courtesy of Prof

R. Ewers)

Algipore is derived from calcifying marine algae, Corallina officinalis. Through a heating process, the algae is segmented in to calcium carbonate and after further refinement processes, part of the calcium carbonate transforming into fluoro hydroxyapatite (Schopper et al., 2003). Algipore has been used as a bone grafting material in sinus augmentation (Ewers, 2005) and periimplantitis treatments (Roos-Jansaker et al., 2011).

Isolation and culture of cells Human periodontal ligament cells were used for Neutral Red Uptake Assay and Dyract AP direct-contact experiments. Bone marrow stromal cells were used for analysing cell differentiation and bone matrix mineralisation in vitro.

Human periodontal ligament (PDL) cells (Paper II)

The usage of human PDL cells was approved by the Ethical Board of Umeå

University. PDL cells were obtained from teeth with no local pathology, extracted from young individuals for orthodontic reasons. After careful rinsing, PDL cells were isolated from the roots by bacterial collagenase digestion (Ransjo et al. 1999). Released PDL cells were collected and cultured in a-MEM with serum and antibiotics. The cells were grown at 37 ̊C in a humidified atmosphere of 5% CO2 in air. Cells from passages 3-9 were used for the experiment.

Bone Marrow Stromal Cells (BMC) (Paper II, III)

The protocol for usage of mice was approved by the Animal Care and Use Committee of the University of Umeå. Bone marrow cells (BMC) were isolated from 6–9 weeks old male CsA mice. The tibiae and femur were removed and dissected free under sterile conditions from adhering soft tissues. The bone ends were cut off to access the marrow cavity and the bone marrow was flushed out with α-MEM medium. BMC were collected and cultured at 37 ̊C in a humidified atmosphere of 5% CO2 in air (Ransjo et al., 1999). BMC were used for analysing the effects of DAP

24

on gene expression and for analysing bone nodule formation (Paper I) and for effects of Bio-Oss on growth pattern and nodule formation (Paper II).

Rat Calvarial Cells (RC) (Paper III)

Rat calvarial (RC) cells were isolated from calvaria of decapitated 2 day old Sprague Dawley rats, according to a modification of the technique described earlier (Irie et al., 1998). The calvaria were dissected under aseptical conditions and cells were extracted using a sterile enzymatic digestion solution. The solution was changed after 5, 10 and 15 minutes and discarded after each time. Remaining cells after the 4th enzymatic treatment were cultured to confluence.

Osteoblastic mouse cell line (MC3T3-E1) (Paper III)

MC3T3-E1 is a mouse osteoblastic-like cell line (DSMZ GmbH Germany). The cells were cultured in complete cell culture media and the culture media were changed every 2-3 days.

DAP procedures

Preparation of DAP specimens (Paper II)

One capsule of DAP colour A3 was moulded between two Thermanox cover slips (13 mm diameter) and light cured for 60 seconds with an light intensity above 400mW/cm2 with 13-mm light probe (Spectrum Curing Light, Dentsply). Specimens were made in accordance to ISO 10993-5:1999, for in vitro cytotoxicity test. The cover slips were removed and the edges of the DAP specimens were carefully washed with phosphate buffered saline in order to remove any unpolymerized material.

Medium extract of DAP (Paper II)

Specimens of DAP were placed in α-MEM with 10% FCS, antibiotics and incubated at 37◦ C for 7 days. The 100 % DAP extract defined as one specimen prepared from 25 mg DAP placed in 2ml α-MEM. 100% DAP extract was diluted with α-MEM culture media (+10% FCS) to obtain 10, 1, 0.1 and 0.01% concentrations of DAP extract.

DAP direct-contact tests (Paper II)

Human PDL cells were seeded onto or around a DAP specimen and cultured in 35 mm culture dishes for 6 days. Care was taken not to dislodge the specimen from the bottom of the culture dish during the culture period. The control group consisted of PDL cells cultured under same condition but without any DAP specimens. After culture, the test and control groups were fixed in 4% formalin and stained with toluidine blue. Morphological evaluation of cell morphology and growth pattern on

25

and around the DAP specimens was performed. Microphotographs of representative areas were taken.

Analysis of number of viable cells Neutral red uptake is linear to cell number as confirmed with cell count and protein determination by (Borenfreund and Puerner, 1985)

Neutral Red Uptake Assay (Paper II)

In order to assess the number of viable and intact cells, Neutral Red (NR) Assay was performed. Viable cells will take up the red NR dye by active transport and incorporate the dye into lysosomes, whereas non-viable cells will not take up the dye. NR uptake analysis was performed in accordance with the method described earlier (Borenfreund and Puerner, 1985, Repetto et al., 2008). Human PDL cells were seeded in 96-well micro plates and cultured for three days, with or without different concentrations of DAP extract. Cells were then incubated with the NR dye in α-MEM culture medium in 37 ̊C for 3 hours. The cells were fixed with formalin and CaCl2 and thereafter treated with alcohol and acetic acid to release the NR dye taken up by the cells. The concentration of dye was analysed in a spectrophotometer to assess the effect of DAP extract on cell viability as compared to control cultures.

Gene expression analysis Effects of DAP extract on BMC differentiation were determined by analysing the gene expression for osteoblasts markers.

RNA extraction and cDNA synthesis (Paper II)

Total RNA was extracted, purity and quantity of RNA was determined and RNA was treated with deoxyribonuclease I. Thereafter RNA was reverse transcribed into single-stranded cDNA, which was kept at −20 ◦C until used for quantitative real-time PCR.

Quantitative real-time PCR (Paper II)

Gene expression was analysed using quantitative PCR and specific primers and labelled probes. The relative mRNA expression for α-1procollagen, alkaline phosphatase (ALP), osteocalcin was computed using the standard curve method and compared to the expression for β-actin.

Bone nodule mineralization assay Effects of DAP and Bio-Oss extracts on “bone formation” in vitro were analyzed in BMC, rat calvarie (RC) cells and an osteoblast mouse cell line (MC3T3-E1) with a mineralization bone nodule assay. Cell cultures were fixed and stained for calcium and phosphate using Alizarin Red and von Kossa staining before the assessment of

26

nodule formation. Cells were cultured in α-MEM medium without or with different concentrations of DAP and Bio-Oss medium extracts. Ascorbic acid and β-glycerophosphate were added to the culture media to facilitate the mineralization process and formation of bone nodules (Bellows et al., 1986). The cells were cultured for 3-4 weeks and thereafter fixed and stained with either Alizarin Red or Von Kossa.

Von Kossa staining (Paper II and III)

AgNO3 was applied to the fixed cell cultures, which thereafter were exposed to bright light/ UV light for 60 minutes (Bonewald et al., 2003). After thorough rinsing with PSB, cell cultures were dried and nodule formation assessed under a microscope. Black stained clusters with more than 10 cells were defined as a nodule. Microphotographs of representative areas were taken.

Alizarine Red staining (Paper III)

Alizarine red staining dye solution was adjusted for pH and added to cell cultures and incubated at room temperature for 30 minutes (Gregory et al., 2004). Cell cultures were rinsed to remove excess dye. Microphotographs of representative areas were taken.

Elementary ion release analysis

Fluoride ion release (Paper II)

Fluoride concentration of DAP 100% extract was analysed according to approved ASTM method (ASTM D 1179) with Orion Research 901 microprocessor ion analyser and Orion ionplus Fluoride Electrode 96-09.

Inductively coupled plasma optical emission spectroscopy, ICP-OES

(Paper II, III, IV)

The medium extract was analysed with regards to the content of elementary ions, Ca, P, Si and Na. The extract was introduced in to the ICP ionising torch of Argon gas. The light emitted by the atoms or ions was collected by a monochromator and resolved into spectral lines which were detected by a spectrometer. A quantitative analysis of the concentration of elements in solution was computed by comparison with a standard solution.

Medium extract of bone graft materials

Extract Bio-Oss, Bio active glass 45S5, Algipore (Paper III, IV)

Particles of the test materials, BioOss, bioactive glass (45S5) and Algipore were immersed in α-MEM culture media (1% w/v) with or without 10% FCS, for different

27

times, at 37 ̊C. The extract was analysed using ICP-OES with regards to content of silicon, calcium, phosphate and sodium.

Surface analysis of bone graft substitutes

Surface properties of the bone graft substitutes, bioactive glass (45S5), Bio-Oss, Algipore were analyzed to evaluate precipitation, dissolution, chemical composition and surface charge. The bone graft substitutes were exposed, at different times, to α-MEM with/without serum. SEM/EDAX was used to analyze the bone graft surface topography and elementary composition of the bone graft substitutes. Precipitation/dissolution was analysed with ICP-OES technique and Calcium-45 isotope uptake.

Scanning Electron Microscopy/Energy Dispersive X-ray analysis

(SEM/EDAX)(Paper III, IV)

Bone graft substitutes were incubated α-MEM for different times. The washed and dried biomaterials were analyzed, without surface coating, with regards to surface topography and chemical composition with SEM/EDAX

Cryogenic X-ray Photoelectron Spectroscopy fast frozen samples (Cryo-

XPS) (Paper IV)

Cryogenic X-ray photoelectron spectroscopy (Cryo-XPS) was used to analyse the solid- liquid interface of bone graft substitutes in cell culture media with regards to chemical and physical composition and processes. The bone graft substitutes were separated from the cell culture media by removing the liquid, leaving a paste for XPS analysis. The pastes of biomaterials were instantly frozen with liquid nitrogen and analysed with XPS in a ultrahigh vacuum. The XPS spectra were obtained and electrons from Al Kα analysed. The binding energy of the analysed surface is specific for each element. The binding energies were recorded and referred to C 1s line of aliphatic carbon.

Calcium 45 isotope labeling (Paper III, IV)

Bio-Oss particles were incubated (24h) with α-MEM culture media and added 45Ca isotope to analyze Ca uptake to the surface. Bio-Oss particles were separated from the incubation media and dissolved in hydrochloric acid. 45Ca isotope in the media and in the dissolved BO particles was analyzed with a Liquid Scintillation System and calcium uptake was calculated.

28

Results and discussion

Paper I, II The properties of the root end filling material are of great importance for

the healing results of root end operations. Biocompatibility, manageability and sealing properties of the material are considerations influencing the choice of the material and operation technique. In Paper I, the materials Dyract AP with Prime&Bond NT, were used with a modified operation technique, and evaluated as an alternative for the commonly applied, but moisture sensitive, glass ionomer Ketac Silver in root end surgery.

Apical healing after 1 year was significantly better for the DAP group when compared with the glass ionomer group, according to Molven PAI score. In the DAP group 79 % of the periapical infections healed completely, and including scar tissue, 89 % healed satisfactorily. For the glass ionomer group only 44% healed satisfactorily, the remaining lesions were diagnosed as failed or uncertain healing (Tab 1).

Table 1. Healing result for Dyract AP/Prime&Bond NT and Ketac Silver

when used for root end surgery in single rooted teeth with persistent

periapical infection.

The sealing capacity of the root end material is of importance. Glass

ionomer is sensitive to early moisture exposure during initial setting time (McKinney et al., 1987, Khouw-Liu et al., 1999). Bleeding and saliva contamination will impair the sealing properties of the material and probably explain the high failure rate for Ketac Silver in the present study. Other reasons for failures could be that the teeth included in the study were technically difficult with inadequate root fillings, and could have undetected cracks and accessorial canals, which impair healing results for glass ionomers with the technique recommended. DAP gives a better possibility for good sealing due to the technique with a bonding system. In addition light curing gives a faster and more controlled curing, thus preventing moisture contamination. The root end preparation technique probably also influenced the healing result in the present study. Shorting of the root apex

29

exposes the apical delta of root canals exit bacterial and bacterial toxins. Furthermore the root end preparation with a cylinder used for Ketac Silver might impair the remaining length of the root filling. The shallow preparation used for the light cured bonded DAP sealed the whole of the denuded root thus reducing leakage and promoting healing. As can be seen in Table 1, teeth with previously inadequate placed root canal filling have a higher failure rate in the Ketac Silver group in comparison to the DAP P&B NT group. The good healing results for DAP with P&B NT are similar to that of Retroplast (Rud et al., 1996) having similar operation techniques and using a bonding system. This means that the complete coverage of the denuded dentine surface with a bonded compomer increases the apical healing as compared to a non-bonded material. Other bonded composites might function well as an alternative but the material properties such as, dentine bonding and composition of the filler particles, might have an effect of the healing result. Interestingly, the filler particles of DAP are a strontium-fluoro-silicate glass. Strontium and fluoride ions have been shown to be released from DAP and release dependent on the solution and pH (Sales et al., 2003). Strontium ranelate and fluoride have been used for the treatment of bone loss in osteoporosis patients (Ammann, 2005, Guañabens et al., 2000). Silicon is also an ion which might be released from the glass filler particles in the DAP. Silicon is suggested to have beneficial effect on bone healing (Jugdaohsingh, 2007). This implies that the release of silicon, strontium and/or fluoride might have an effect on the apical bone healing when DAP is used as a root end filling material. Reoperation of teeth with DAP and Ketac Silver was performed on the teeth with failed healing. Loose root end fillings were the cause of the failures in the DAP group and also the cause of failures for the Retroplast method (Rud et al., 1997). It is of utmost importance that the material is properly handled. Curing must be performed according to the manufacturer’s instructions, otherwise unlinked monomers and additives from the resin may leak (Van Landuyt et al., 2011) and could cause toxic reactions. In a histological study on rat implanted Dyract, specimens produced a moderate inflammation that decreased with time (Ozbas et al., 2003). In the Ketac Silver group there was no obvious cases with poor adhesion as a cause for failure, but the surface of the material appeared, in some cases, to be porous. To further investigate the biological effect of DAP and ion release an experimental study was performed (Paper II). PDL cells cultured on and around DAP specimens showed similar growth pattern and morphology as cell cultured on cover slips (Thermanox) treated for enhanced cell attachment and growth. This demonstrates that DAP is biocompatible when adequately light cured. The cells used for the contact test, human periodontal ligament (PDL) cells, are highly relevant as they are the cells in contact with the DAP retrograde filling in vivo.

30

Under-curing of the resin containing material is demonstrated to have a toxic effect on cells (Quinlan et al., 2002) (Ergun et al., 2011). The importance of adequate exposure time with light curing techniques is demonstrated with DAP specimens inadequately light cured, (20 sec; 50 % of manufacturers recommended curing time). The cell free zone surrounding the under-cured DAP specimens indicate that toxic products are leaching from DAP and thus could have negative effects on the healing results in patients (Fig. 16). When analysing soluble products (dissolution products/eluates/extract) from biomaterials/dental materials mostly ethanol and distilled water are used as dissolution fluids (Michelsen et al., 2003, Geurtsen, 2000, Ortengren, 2000, Ortengren, 2001, Ferracane and Condon, 1990). In the present studies cell culture medium was used as an extract fluid since it is more comparable to the in vivo situation and furthermore enables the use of extract in cell culture assays. Viability and growth of human PDL cells were quantified for different concentrations of DAP extract prepared from optimally cured DAP specimens incubated in cell culture medium. No significant differences in cell numbers were obtained comparing cells cultured with the highest concentration of DAP extract to control cultures. Low concentrations of the DAP extract had a stimulatory effect on cell growth. This inversed concentration dependence of DAP extract on cell growth can only be speculated upon. It may be related to different products in the extract, some having stimulatory whilst others may have inhibitory effects on cell growth.

(a) (b) (c)

Fig.16. Human PDL cells cultured in contact with adequate (a), inadequate

light cured (b) and (c) on the surface of an adequately cured DAP specimen

In contrast to the results in paper II, a moderate toxic effect of Dyract AP on human pulp fibroblast has been reported in a latter study (TunÇ et al., 2009). The Dyract AP specimen was placed on an agar-culture medium

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(DMEM) system agar-DMEM with the cells on the bottom of the culture well. The concentration dependence of DAP extract could thereby be evaluated. However the result from the present study and the study by TunÇ et al. may not fully reflect the clinical situation (described in Paper I) since there is a constant exchange of tissue fluid round the root-end filling diluting the dissolution products from the DAP. The release of Si and Sr from Dyract filler particles (1% w/v) incubated in α-MEM cell culture medium was analysed with ICP-OES and the concentrations Sr was 21.9 ±0.7 ppm and silicon 16.9 ± 0.14 ppm (data not shown). Strontium has positive effects on bone remodelling and strontium ranelate used for treatment of osteoporosis patients (Marie et al., 2010). Furthermore silicon is suggested to have positive effects on bone healing (Jugdaohsingh, 2007). Together the release of strontium and silicon ions from the filler glass particles initiated a further analysis of ions in the extract from light cured DAP specimens with ICP-OES (Paper II). Compared to the glass filler particles the DAP specimens released lower levels of Sr (15 ppm) and Si (1.5) as the resin material in the DAP specimens are bound to the filler particles are bound to silanezed filler particles thus reducing strontium and silicon release. PDL cell proliferation increase in all concentrations of DAP extract. Mouse BMC expression of ALP and osteocalcin in DAP extract concentration of 1, 10, 100% and a decrease in bone nodules formation for 10 and 100% DAP extract. The result demonstrates a stimulatory effect on cell growth and no toxicity. The reduced expression of osteoblastic markers might be explained by a longer stage of proliferation in the higher concentrations. The concentration of Sr leaked from DAP is too low to have an effect on general bone remodelling, however concentration of 21 ppm induces expression of osteogenic markers in a human bone marrow mesenchymal stem cells (Sila-Asna et al., 2007). Sr released from DAP root end filling could reach a higher concentration in the proximity of the root apex and may have an effect on local bone healing. F concentration (0.2mM) in the DAP extract was low but might still have an effect on gene expression, as indicated by a study demonstration an inhibitory effect on osteocalcin and type 1 collagen mRNA expression (Li and DenBesten, 1993).

Paper III, IV

Biomaterials are frequently used in dento-alveolar surgery as bone graft substitutes in treatment of bone defects. Bio-Oss, Bioactive glass (45S5) and Algipore are commercially available and are commonly used for bone replacement after bone loss has arisen from pathological processes (Roos-Jansaker et al., 2011, Scheyer et al., 2002, Sculean et al., 2004). The clinical

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outcome of the surgery is sometimes difficult to predict since the interactions between the biomaterial and the surrounding tissue are largely unknown.

The Bio-Oss particles close contact to the newly formed bone, as seen in biopsies, raises questions about whether or not the biomaterial has properties which may influence bone formation. Very little is known about Bio-Oss surface interaction with the surrounding biological fluids and tissues. The first analysis was therefore to examine possible ion exchange/release from the Bio-Oss particles.

Simulated body fluid (SBF) is often used as an incubation medium when studying biomaterial dissolution/precipitation reactions (Al-Munajjed et al., 2009, Blaker et al., 2011). The validity of the results obtained in SBF when compared to the in vivo situation has come in to question since SBF is lacking essentials such as proteins (Bohner and Lemaitre, 2009). To better mimic the biological environment and the surface reactions of Bio-Oss, the present studies are using α-MEM as the incubation medium. The usage of a cell culture medium also gives a better correlation between the surface precipitation /dissolution reactions and the analyzed interactions between Bio-Oss particles and cell cultures.

ICP-OES analysis of the Bio-Oss incubation medium (extract) show significantly reduced Ca and P levels after 24 hours in comparison to the control medium. The effect of Bio-Oss on reduction of Ca and P in the incubation media was dose dependent and seen at very low concentrations. A significant reduction of Ca and P was even seen with 0.1 v/w% Bio-Oss, corresponding to 10 granulates/ml of incubation medium. Adsorption of Ca and P onto the surface of the Bio-Oss particles caused a decrease in these ions in the Bio-Oss incubation medium. The uptake of ions was confirmed with a calcium-45 isotope labeling technique (paper III), thus demonstrating that the ions are precipitated on to the Bio-Oss surface. The findings are important when implementing methods for evaluating Bio-Oss in experimental studies. The results were taken in to consideration when designing cell culture studies using Bio-Oss particles. A too high Bio-Oss/media ratio may produce faulty results, due to Ca and P deficiency in the media. The significant reduction of calcium in the cell culture media, below physiological levels might explain some earlier results, reporting that Bio-Oss cause cell death (Beloti et al., 2008, Herten et al., 2009). The concentration of Bio-Oss particles, in relation to the cell culture media used in experimental studies, is seldom reported, thereby restricting the evaluation of the possible effect on cell growth due to decreased calcium levels. In the clinical situation, Bio-Oss may impair healing when densely packed in a confined area, where there is a poor exchange of fluids.

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An interesting new finding was that silicon (Si) could be detected in the medium extract from Bio-Oss. The levels of Si in different batches of Bio-Oss varied considerably (paper III). It might not be surprising to detect Si in Bio-Oss extract, since Si is a trace element in bone tissue (Seaborn and Nielsen, 2002). Si is indicated to be important for skeletal development (Carlisle, 1972) and suggested to play an important role in collagen synthesis and osteoblast differentiation (Reffitt et al., 2003). Si is a readily occurring element in soil, naturally present in plants and made biologically available as orthosilicic acid in the diet (Jugdaohsingh, 2007). As Bio-Oss is a naturally derived (bovine) biomaterial it might be speculated that the difference in Si levels in different batches might be due to an inconsistency of Si content in the animal food. According to the manufacturer, no standardization of animal feed is stipulated for the livestock intended for Bio-Oss production. Different cell cultures (mouse bone marrow stromal cell, rat calvarie cell and the pre-osteoblastic cell line MC3T3-E1) were used to study the possible effects of Bio-Oss on osteoblast differentiation and the mineralization process in vitro. The different cells were cultured for 28 days with only 10 Bio-Oss particles per ml extract medium to avoid Ca depletion in the medium. The growth pattern was the same for all cell types and showed a condensation of cells around the Bio-Oss particles. An in vitro assay was used to investigate the effects of Bio-Oss particles on bone formation/mineralization. With all three cell types, a condensation of cells was demonstrated around the Bio-Oss particles. A mineralization process in the cell condensations (nodule) was demonstrated with von Kossa (phosphate) and Alizarine Red (calcium) staining. The possible Ca-P precipitation on Bio-Oss particles, as demonstrated with ICP-OES and 45Ca isotope labeling, indicates that Bio-Oss interacts with the biological media. Dissolution/precipitation reactions on Bio-Oss particles were therefore further analyzed (paper IV).

In addition to Bio-Oss, the solid-solution interface of two biomaterials commercially available and used in dento-alveolar surgery as bone graft substitutes, Bioactive glass (45S5) and Algipore, were also analyzed. Bioactive glass (45S5), a synthetic biomaterial containing Si, was used as a reference material since the chemical composition and reactions have been extensively investigated (Hench et al., 2004, Jones et al., 2001, Sepulveda et al., 2002a, Xynos et al., 2000). α-MEM cell culture media, with or without added serum, was used as a solution, in which biomaterials were placed. The morphological analysis of the biomaterials by SEM showed Bioactive glass 45S5 “as received” to have a rough surface structure that was smoothed when the surface was exposed to the culture medium. No morphological surface changes were seen for Bio-Oss or Algipore when exposed to the medium.

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Dissolution in the culture media of ions from the biomaterials (1%v/w) was analyzed with ICP-OES. In Bioactive glass, the major surface reactions take place in the first 24 hours. Ca concentration in the bioactive glass extract (1 % w/v) showed a significant increase with a peak in the first few hours before returning to original culture medium levels after 24 hours. No further changes in Ca concentration were detected over time. The concentrations of P in the medium was reduced to 50 % after 24 hours and remained at that level for 168 hours. Significantly high levels of Si were detected in the extract within the first 24 hours. Our results of the dissolution/precipitation reactions after 24 hours are confirming earlier published studies (Sepulveda et al., 2002a). After the first 24 to 72 hours significant reductions of Ca and P levels in the Bio-Oss and Algipore extract were demonstrated. The Ca dissolution/precipitation in Bioactive glass (45S5), Bio-Oss and Algipore was confirmed by 45Ca isotope labeling. Free isotope in the media was reduced and instead detected at the surface of the biomaterial indicating an adsorption with biomaterial affinity for calcium. Ca concentrations on the biomaterial surfaces increased slightly in Bio-Oss as serum were added. The significant reduction of Ca levels in culture media even at low biomaterial/culture media ratios has effects on study designs for evaluating cellular/biomaterial interactions. The choice of solution is important when analyzing dissolution/precipitation reactions of biomaterials. SBF is often used when studying calcium phosphate apatite formation as a surface reaction on biomaterials (Kokubo and Takadama, 2006, Jalota et al., 2006, Al-Munajjed et al., 2009) but lacks in proteins. The effect of added serum has implication on surface reaction of bone graft substitutes such as corrosion process of Bioactive glass (El-Ghannam et al., 2001, Radin and Ducheyne, 1996).

Elemental composition of the material surface was analysed using EDAX, allowing a Ca/P ratio to be calculated. The Ca/P ratio was used as an indication of adsorption/dissolution processes on the surface. The Ca/P ratio for Bioactive glass (45S5) decreased day one while Bio-Oss and Algipore had an unchanged Ca/P ratio as compared to the ratio of the material “as received”. Understanding the chemical composition and chemical reactions in the boundery of the solid biomaterial and the surounding solution is very important in gaining an understanding of the function of the biomaterials. EDAX measures the chemical composition in the range of a few microns and thereby not strictly analysing the surface, but also the underlying bulk of the biomaterial. XPS analysis measuring at the nano level was therefore performed on wet samples of the biomaterials in α-MEM culture medium, with and without serum. Cryo-XPS fast frozen sample technique has made it

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possible to analyse the solid-solution interface without altering the solid-solution interface (Shchukarev, 2006).

A decrease in the surface Ca/P ratio for Bioactive glass 45S5 in the first 24 hours was demonstrated with Cryo-XPS (fast frozen technique) after incubation in culture media and an equal ratio was seen after 7 days. With added serum in the incubation media, Ca/P ratio for Bioactive glass (45S5) decreased compared to the ratio for day one without serum. After 7 days the Ca/P ratio could not be determined, instead, high levels of nitrogen (N) were measured, indicating that the biomaterial surface is covered by a protein layer. There were only small changes in the surface (Bradley et al., 2004) Ca/P ratio for Bio-Oss and Algipore after incubation in culture media with or without serum after 1 to 7 days. The surface charge detected with XPS, as Na/Cl ratio, showed an interesting change for Bio-Oss when serum was added. The Bio-Oss surface was negative in α-MEM but changed to positive as serum was added to the medium. Cryo-XPS data indicated, by the presence of C and N, an adsorption of proteins to the surfaces of Bio-Oss, Bioactive glass (45S5) and Algipore when the materials were incubated in culture media with serum.

Surface reactions and protein adsorption on the biomaterials Bio-Oss, Bioactive glass (45S5) and Algipore may be important for cell adherence, cell functions and new bone formation.

General Discussion

Implanting a biomaterial in a patient during an operation requires not only surgical skills, but also knowledge of the biomaterial. Biomaterials used in dento-alveolar surgery are classified as medically technical products, the full content of the material does not have to be reported. Is the biomaterial therefore considered inert? Dental materials have been used in root end surgery without specifically being developed for this purpose, but have still shown good healing results. Dental materials developed for treating tooth decay, have limitations in the dento-alveolar surgical situation, since their properties might render the material less suitable due to blood contamination. A material, which has properties such as providing a tight seal with a short curing time, is therefore required. A light cured compomer was investigated as an alternative to a commonly used glass ionomer. The compomer also required an alternative operation technique with a shallow saucer shaped root end preparation. The healing result was significantly better than that obtained with a conventional root end operation. By

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covering the whole of the denuded root surface with the bonded compomer it was possible to cover the exposed dentine tubule, thus preventing any further leakage of bacterial products. According to the product labeling, the filler particles of the compomer contained strontium, which has been added to bioactive glass in order to increase osteoblast and reduce osteoclast function (Gentleman et al., 2010). Strontium is used in osteoporosis treatment, as the element inducing bone formation and reducing bone resorption (Kanis et al., 2011). The effect of strontium in the filler particles might be a factor in the apical bone healing. Ions of Sr, Si and F leaching from the compomer and the filler particles were analyzed. The levels of Sr was below the reported therapeutic levels for strontium ranelate, but since the ion concentrations might be sufficiently high nearest to the PDL cells in the parodontium apically and possibly have an effect on bone healing.

Bioactive glass release ions of Si, Ca and P effecting bone formation. There has been little investigation of ion release from non-synthetic bone graft substitutes. Naturally occurring bone graft substitutes, derived from bovine bone and marine algae, are commercially readily available. Histological studies have shown that bone forms closely to these biomaterials. The interface between bone and biomaterial is largely unknown. Surface topography is described by the manufacturers, but the solid-solution interface formed as the biomaterial is implanted during the surgical procedure has not been investigated. This solid-solution interface is the connection surface between the bone cells and the biomaterial. Ion dissolution from the surface of the bone graft substitutes was shown. Adsorption, dose dependent, of vital ions from the culture media effecting cell proliferation/differentiation was detected in one of the bone grafts. This has implications as to how in vitro studies are to be designed, since false negative results might be obtained. The alterations in surface charges and protein adsorption on the surface of the bone graft substitutes need further investigations shedding light on the role of the interface in bone formation A deeper understanding of the chemical processes, by analyzing the solid-solution interface with different techniques at micro-and nano levels, is therefore a part of understanding the difference between biomaterials with regards to morphology, chemical composition, ion exchanges, surface charges, and protein adsorption. The knowledge of the surface reactions and the solid-solution interface is important in predicting the bone healing outcome, when using biomaterials in the clinical work.

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Conclusion

General Conclusion

It is important to understand the surface reactions of the biomaterials used in dental surgery as these reactions of the materials could have profound effects of bone healing when used in regenerative surgery.

Specific Conclusions

� A light cured compomer with a dental adhesive in an adjusted operation technique significantly increased healing outcome in root end surgery as compared to a conventional root end operation using a glass ionomer material. The healing result might be due to achieving a tight seal over the whole of the exposed denuded root surface and/or could be due to biomaterial surface effects (Paper I).

� Cell displayed normal morphology and growth pattern in close contact with the compomer. Medium extract of the compomer increased PDL cell proliferation but decreased bone mineralization in bone marrow cultures. Inorganic ions, known to have osteogenic properties, strontium, silicon and fluoride, are dissolved from glass filler particles in the compomer. The concentrations reached in this experimental set up are too low to have a general affect on bone healing. However the concentration levels might be higher close to the material when used as a root end filling in the patient (Paper II).

� Dissolution/precipitation reactions occur at the surface of deproteinized bovine biomaterial (Bio-Oss) leading to an uptake of Ca and P to the surface of Bio-Oss in dose dependent manner. Release of Silicon from Bio-Oss was detected and the amount differed between the biomaterial batches. Bone mineralization in vitro was seen in close contact with Bio-Oss particles in cell cultures. Bio-Oss lowers concentrations of Ca and P in the biological media which can inhibit cell growth and this have implications for design of cell experiments and may also affect the clinical treatment outcome (Paper III).

� Surface dissolution/precipitation reactions occur for all three biomaterials when incubated in a biological medium in both the absence and presence of serum proteins. The biomaterial acquired a surface charge when incubated in the biological media and proteins were

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absorbed on to the surface when the biomaterials were exposed to serum. The solid-solution interface of the bone graft substitute materials will presumably be of importance for the biological response when used for regenerative surgery (Paper IV).

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Acknowledgements So it is done! The thesis is here in front of me ready to push the “send button” on my pc to the printers, thinking of how I can, well enough, express my deepest gratitude to all of you in the “Acknowledgements” I would like to say a warmest “thank you” to all of, my tutors, colleagues, friends and family. I´m so grateful to you all!

Especially thank you to: My supervisor Assoc. professor Maria Ransjö; for believing in me, for giving me the opportunity to convert my ideas about biomaterials into this project, for showing endless patience (and impatience when I needed it) “beyond the call of duty” and for giving me an insight in to your deep knowledge of bone and the fascinating world of bone research. Thank you!

To my co-supervisor and co-author Professor Ulf Örtengren; thank you for allowing me to draw heavily on your knowledge and for the stimulating and fruitful discussions regarding this project and for giving me good advice about life as a PhD student

Dr Britta Willman (Andersson) thank you for great support and help

Dr Ian Thompson; Thank you for giving me the opportunity to visit your laboratory and for sharing your knowledge of Bioactive glass. It was fun making bioactive glass! Thank you

Dr Karin Wannfors; for sharing your knowledge and surgical skills.

To my co-authors; Assoc.professor Martin Andersson, Assoc.professor Erik Björn and Assoc.professor Andrey Shchukarev, thank you for sharing your knowledge and giving me a deeper insight to the micro-nano world.

A very warm and special thank you to my co-author, co-PhD student and friend Živko Mladenović. Thank you for all your help, support, and willingness to share your knowledge of EndNote, Word, articles, Powerpoint and the fastest way to PerkinElmer. Thank you for your work and input to the project, It´s been a privilege and we had some fun.

Dr Rolf Claesson for providing facilities for fluoride analysis and always being encouraging.

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Professor Gunnar Warfinger for working miracles with the biopsies I sent to you in Malmö and I got fantastic photographs in return

Anita Lie for helping me with laboratory work on the project.

Professor Ulf Lerhner for his generous support.

Assoc Professor Pernilla Lundberg Assoc. Professor Anders Johansson and Professor Jan van Dyken; for talking, caring, advising and encouraging .

To my friend and colleague Dr Margareta Fredriksson for endless support .

Lillemor Hägglund for helping me, reminding me and being very understanding when I had forgotten to do what I was supposed to do.

I would also like to thank my colleagues at the laboratory for endless support and good advice; Ingrid Boström, Inger Lundgren, Chrissie Roth, Birgit Andertun and Maj-Britt Edlund: Thank you for cheering me along. It was great working with you all!

To former PhD students; Georgios Belibasakis, Emma Persson, Stephen Matemba, Py Palmqvist, Anna Brechter, Susanna Granholm, Peyman Kelk; It was great to work with you.

Present PhD students; Ali Kassem, Fredrik Strålberg, Cecilia Koskinen and it´s great to work with you, chat with you and have a good laugh

To all my wonderful colleagues, who are also my friends, in both Sweden and Norway, who have so patiently waited for me to come back to work; you have all been so supportive and helpful. Thank you!

A very special thank you to my dental nurses, past and present, for rearranging my time book, making it possible to see this thesis through, thank you!

To my family; I`m back! (Promise!)

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References

Al-Munajjed, A. A., Plunkett, N. A., Gleeson, J. P., Weber, T., Jungreuthmayer, C., Levingstone, T., Hammer, J. & O'Brien, F. J. (2009) Development of a biomimetic collagen-hydroxyapatite scaffold for bone tissue engineering using a SBF immersion technique. J Biomed Mater Res B

Appl Biomater 90, 584-591.

Ammann, P. (2005) Strontium ranelate: a novel mode of action leading to renewed bone quality. Osteoporos Int 16 Suppl 1, S11-15.

Allori, A. C., Sailon, A. M. & Warren, S. M. (2008a) Biological basis of bone formation, remodeling, and repair-part I: biochemical signaling molecules. Tissue Eng Part B Rev 14, 259-273.

Allori, A. C., Sailon, A. M. & Warren, S. M. (2008b) Biological basis of bone formation, remodeling, and repair-part II: extracellular matrix. Tissue Eng Part B Rev 14, 275-283.

Araujo, M. G. & Lindhe, J. (2005) Dimensional ridge alterations following tooth extraction. An experimental study in the dog. J Clin Periodontol 32, 212-218. doi:CPE642

Askmyr, M., Sims, N. A., Martin, T. J. & Purton, L. E. (2009) What is the true nature of the osteoblastic hematopoietic stem cell niche? Trends

Endocrinol Metab 20, 303-309.

Baldini, N., De Sanctis, M. & Ferrari, M. (2011) Deproteinized bovine bone in periodontal and implant surgery. Dental Materials 27, 61-70.

Barrere, F., van Blitterswijk, C. A. & de Groot, K. (2006) Bone regeneration: molecular and cellular interactions with calcium phosphate ceramics. Int J Nanomedicine 1, 317-332.

Bellows, C. G., Aubin, J. E., Heersche, J. N. & Antosz, M. E. (1986) Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. Calcif Tissue Int 38, 143-154.

Beloti, M. M., Martins, W., Xavier, S. P. & Rosa, A. L. (2008) In vitro osteogenesis induced by cells derived from sites submitted to sinus grafting with anorganic bovine bone. Clinical Oral Implants Research 19, 48-54.

42

Blaker, J. J., Nazhat, S. N., Maquet, V. & Boccaccini, A. R. (2011) Long-term in vitro degradation of PDLLA/bioglass bone scaffolds in acellular simulated body fluid. Acta Biomater 7, 829-840.

Bohner, M. & Lemaitre, J. (2009) Can bioactivity be tested in vitro with SBF solution? Biomaterials 30, 2175-2179.

Bonewald, L. F., Harris, S. E., Rosser, J., Dallas, M. R., Dallas, S. L., Camacho, N. P., Boyan, B. & Boskey, A. (2003) von Kossa staining alone is not sufficient to confirm that mineralization in vitro represents bone formation. Calcif Tissue Int 72, 537-547.

Bonewald, L. F. & Johnson, M. L. (2008) Osteocytes, mechanosensing and Wnt signaling. Bone 42, 606-615.

Borenfreund, E. & Puerner, J. A. (1985) Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol Lett 24, 119-124. Buckwalter, J.A, Glimcher M.J., Cooper, R.R, Recker, R. (1996) Bone Biology, Part 1: Strukture, Blood Supply, Cells, Matrix and mineralization.J Bone Joint Surg 77-A:1256-1275

Cardaropoli, G., Araujo, M. & Lindhe, J. (2003) Dynamics of bone tissue formation in tooth extraction sites. An experimental study in dogs. J Clin Periodontol 30, 809-818.

Carlisle, E. M. (1972) Silicon: an essential element for the chick. Science 178, 619-621.

Civitelli, R. (2008) Cell-cell communication in the osteoblast/osteocyte lineage. Arch Biochem Biophys 473, 188-192.

Cohen, M. M., Jr. (2006) The new bone biology: pathologic, molecular, and clinical correlates. Am J Med Genet A 140, 2646-2706.

Dahlin, C. & Johansson, A. (2010) Iliac Crest Autogenous Bone Graft versus Alloplastic Graft and Guided Bone Regeneration in the Reconstruction of Atrophic Maxillae: A 5-Year Retrospective Study on Cost-Effectiveness and Clinical Outcome. Clin Implant Dent Relat Res.

Davies, J. E. (2007) Bone bonding at natural and biomaterial surfaces. Biomaterials 28, 5058-67

43

Dimitriou, R., Mataliotakis, G. I., Angoules, A. G., Kanakaris, N. K. & Giannoudis, P. V. (2011) Complications following autologous bone graft harvesting from the iliac crest and using the RIA: A systematic review. Injury.

El-Ghannam, A., Hamazawy, E. & Yehia, A. (2001) Effect of thermal treatment on bioactive glass microstructure, corrosion behavior, zeta potential, and protein adsorption. J Biomed Mater Res 55, 387-395.

Ergun, G., Egilmez, F. & Yilmaz, S. (2011) Effect of reduced exposure times on the cytotoxicity of resin luting cements cured by high-power led. J Appl

Oral Sci 19, 286-292.

Esposito, M., Grusovin, M. G., Coulthard, P. & Worthington, H. V. (2006) The efficacy of various bone augmentation procedures for dental implants: a Cochrane systematic review of randomized controlled clinical trials. Int J

Oral Maxillofac Implants 21, 696-710.

European Society of Endodontololgy (2006) Quality guidelines for endodontic treatment: consensus report of the European Society of Endodontology. International Endodontic Journal 39, 921-930.

Ewers, R. (2005) Maxilla Sinus Grafting With Marine Algae Derived Bone Forming Material: A Clinical Report of Long-Term Results. Journal of Oral and Maxillofacial Surgery 63, 1712-1723.

Ferracane, J. L. & Condon, J. R. (1990) Rate of elution of leachable components from composite. Dent Mater 6, 282-287.

Franz-Odendaal, T. A. (2011) Induction and patterning of intramembranous bone. Front Biosci 17, 3734-3746.

Gazzano, E., Bergandi, L., Riganti, C., Aldieri, E., Doublier, S., Costamagna, C., Bosia, A. & Ghigo, D. (2010) Fluoride effects: the two faces of janus. Curr

Med Chem 17, 2431-2441.

Gentleman, E., Fredholm, Y. C., Jell, G., Lotfibakhshaiesh, N., O'Donnell, M. D., Hill, R. G. & Stevens, M. M. (2010) The effects of strontium-substituted bioactive glasses on osteoblasts and osteoclasts in vitro.

Geurtsen, W. (2000) Biocompatibility of resin-modified filling materials. Crit Rev Oral Biol Med 11, 333-355.

44

Golub, E. E. (2009) Role of matrix vesicles in biomineralization. Biochimica

et Biophysica Acta (BBA) - General Subjects 1790, 1592-1598.

Greer, B. D., West, L. A., Liewehr, F. R. & Pashley, D. H. (2001) Sealing ability of Dyract, Geristore, IRM, and super-EBA as root-end filling materials. J Endod 27, 441-443.

Gregory, C. A., Gunn, W. G., Peister, A. & Prockop, D. J. (2004) An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem 329, 77-84.

Guañabens, N., Farrerons, J., Perez-Edo, L., Monegal, A., Renau, A., Carbonell, J., Roca, M., Torra, M. & Pavesi, M. (2000) Cyclical etidronate versus sodium fluoride in established postmenopausal osteoporosis: a randomized 3 year trial. Bone 27, 123-128.

Hallman, M., Lundgren, S. & Sennerby, L. (2001) Histologic Analysis of Clinical Biopsies Taken 6 Months and 3 Years after Maxillary Sinus Floor Augmentation with 80% Bovine Hydroxyapatite and 20% Autogenous Bone Mixed with Fibrin Glue. Clinical Implant Dentistry and Related Research 3, 87-96.

Hench, L. L. (1998) Bioactive materials: the potential for tissue regeneration. J Biomed Mater Res 41, 511-518.

Hench, L. L., Xynos, I. D. & Polak, J. M. (2004) Bioactive glasses for in situ tissue regeneration. J Biomater Sci Polym Ed 15, 543-562.

Hench, L. L. (2006) The story of Bioglass. J Mater Sci Mater Med 17, 967-978.

Herten, M., Rothamel, D., Schwarz, F., Friesen, K., Koegler, G. & Becker, J. (2009) Surface- and nonsurface-dependent in vitro effects of bone substitutes on cell viability. Clinical Oral Investigations 13, 149-155.

Hoang, Q. Q., Sicheri, F., Howard, A. J. & Yang, D. S. (2003) Bone recognition mechanism of porcine osteocalcin from crystal structure. Nature 425, 977-980.

Hoppe, A., Guldal, N. S. & Boccaccini, A. R. (2011) A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials 32, 2757-2774.

45

Iezzi, G., Degidi, M., Scarano, A., Petrone, G. & Piattelli, A. (2007) Anorganic bone matrix retrieved 14 years after a sinus augmentation procedure: a histologic and histomorphometric evaluation. J Periodontol 78, 2057-2061.

Iezzi, G., Scarano, A., Mangano, C., Cirotti, B. & Piattelli, A. (2008) Histologic results from a human implant retrieved due to fracture 5 years after insertion in a sinus augmented with anorganic bovine bone. J

Periodontol 79, 192-198.

Irie, K., Zalzal, S., Ozawa, H., McKee, M. D. & Nanci, A. (1998) Morphological and immunocytochemical characterization of primary osteogenic cell cultures derived from fetal rat cranial tissue. Anat Rec 252, 554-567.

Jalota, S., Bhaduri, S. B. & Tas, A. C. (2006) Effect of carbonate content and buffer type on calcium phosphate formation in SBF solutions. J Mater Sci Mater Med 17, 697-707.

Jones, J. R., Sepulveda, P. & Hench, L. L. (2001) Dose-dependent behavior of bioactive glass dissolution. J Biomed Mater Res 58, 720-726.

Jugdaohsingh, R. (2007) Silicon and bone health. J Nutr Health Aging 11, 99-110.

Kanis, J. A., Johansson, H., Oden, A. & McCloskey, E. V. (2011) A meta-analysis of the effect of strontium ranelate on the risk of vertebral and non-vertebral fracture in postmenopausal osteoporosis and the interaction with FRAX((R)). Osteoporos Int 22, 2347-2355.

Khouw-Liu, V. H., Anstice, H. M. & Pearson, G. J. (1999) An in vitro investigation of a poly (vinyl phosphonic acid) based cement with four conventional glass-ionomer cements Part 2: Maturation in relation to surface hardness. J Dent 27, 359-365.

Kim, S. & Kratchman, S. (2006) Modern Endodontic Surgery Concepts and Practice: A Review. Journal of Endodontics 32, 601-623.

Kim, Y.-K., Yun, P.-Y., Kim, S.-G., Kim, B.-S. & Ong, J. L. (2009) Evaluation of sinus bone resorption and marginal bone loss after sinus bone grafting and implant placement. Oral Surgery, Oral Medicine, Oral Pathology, Oral

Radiology, and Endodontology 107, e21-e28.

46

Kneser, D. J. S., B. Munder, C. Klemt, C. Andree, & Stark, G. B. (2002) Tissue engineering of bone. Min Invas Ther & Allied Technol 11, 107–116.

Kokubo, T. & Takadama, H. (2006) How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 27, 2907-2915.

Kronenberg, H. M. (2003) Developmental regulation of the growth plate. Nature 423, 332-336.

Lemperle, S. M., Calhoun, C. J., Curran, R. W. & Holmes, R. E. (1998) Bony healing of large cranial and mandibular defects protected from soft-tissue interposition: A comparative study of spontaneous bone regeneration, osteoconduction, and cancellous autografting in dogs. Plast Reconstr Surg 101, 660-672.

Li, R. & DenBesten, P. K. (1993) Expression of bone protein mRNA at physiological fluoride concentrations in rat osteoblast culture. Bone Miner 22, 187-196.

McKinney, J. E., Antonucci, J. M. & Rupp, N. W. (1987) Wear and microhardness of glass-ionomer cements. J Dent Res 66, 1134-1139. Marie, P. J., Felsenberg, D. & Brandi, M. L. (2010) How strontium ranelate, via opposite effects on bone resorption and formation, prevents osteoporosis. Osteoporos Int. 16, Suppl 1, S7-10.

Marsell, R. & Einhorn, T. A. (2011) The biology of fracture healing. Injury 42, 551-555.

Meyer, U., Buchter, A., Wiesmann, H. P., Joos, U. & Jones, D. B. (2005) Basic reactions of osteoblasts on structured material surfaces. Eur Cell

Mater 9, 39-49.

Michelsen, V. B., Lygre, H., Skalevik, R., Tveit, A. B. & Solheim, E. (2003) Identification of organic eluates from four polymer-based dental filling materials. Eur J Oral Sci 111, 263-271.

Mordenfeld, A., Hallman, M., Johansson, C. B. & Albrektsson, T. (2010) Histological and histomorphometrical analyses of biopsies harvested 11 years after maxillary sinus floor augmentation with deproteinized bovine and autogenous bone. Clinical Oral Implants Research 21, 961-970.

Nel, A. E., Madler, L., Velegol, D., Xia, T., Hoek, E. M., Somasundaran, P.,

47

Klaessig, F., Castranova, V. & Thompson, M. (2009) Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater 8,

Nicholson, J. W. & Czarnecka, B. (2004) The release of ions by compomers under neutral and acidic conditions. J Oral Rehabil 31, 665-670.

Orstavik, D. (1988) Reliability of the periapical index scoring system. Scand

J Dent Res 96, 108-111.

Ortengren, U. (2000) On composite resin materials. Degradation, erosion and possible adverse effects in dentists. Swed Dent J Suppl, 1-61.

Ortengren, U., Wellendorf, H., Karlsson, S. & Ruyter, I. E. (2001) Water sorption and solubility of dental composites and identification of monomers released in an aqueous environment. J Oral Rehabil 28, 1106-1115.

Ozbas, H., Yaltirik, M., Bilgic, B. & Issever, H. (2003) Reactions of connective tissue to compomers, composite and amalgam root-end filling materials. Int Endod J 36, 281-287.

Patino, M. G., Neiders, M. E., Andreana, S., Noble, B. & Cohen, R. E. (2002) Collagen: an overview. Implant Dent 11, 280-285.

Quinlan, C. A., Zisterer, D. M., Tipton, K. F. & O'Sullivan, M. I. (2002) In vitro cytotoxicity of a composite resin and compomer. Int Endod J 35, 47-55.

Radin, S. & Ducheyne, P. (1996) Effect of serum proteins on solution-induced surface transformations of bioactive ceramics. J Biomed Mater Res 30, 273-279.

Raggatt, L. J. & Partridge, N. C. (2010) Cellular and molecular mechanisms of bone remodeling. J Biol Chem 285, 25103-25108.

Ransjo, M., Lie, A. & Mackie, E. J. (1999) Cholera toxin and forskolin stimulate formation of osteoclast-like cells in mouse marrow cultures and cultured mouse calvarial bones. Eur J Oral Sci 107, 45-54.

Reffitt, D. M., Ogston, N., Jugdaohsingh, R., Cheung, H. F., Evans, B. A., Thompson, R. P., Powell, J. J. & Hampson, G. N. (2003) Orthosilicic acid stimulates collagen type 1 synthesis and osteoblastic differentiation in human osteoblast-like cells in vitro. Bone 32, 127-135.

48

Repetto, G., del Peso, A. & Zurita, J. L. (2008) Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 3, 1125-1131.

Roos-Jansaker, A. M., Lindahl, C., Persson, G. R. & Renvert, S. (2011) Long-term stability of surgical bone regenerative procedures of peri-implantitis lesions in a prospective case-control study over 3 years. J Clin Periodontol 38, 590-597.

Rud, J., Rud, V. & Munksgaard, E. C. (1996) Retrograde root filling with dentin-bonded modified resin composite. J Endod 22, 477-480.

Rud, J., Rud, V. & Munksgaard, E. C. (1997) Effect of root canal contents on healing of teeth with dentin-bonded resin composite retrograde seal. J

Endod 23, 535-541.

Sarkar, N. K. (1999) Metal-matrix interface in reinforced glass ionomers.Dent Mater 15, 421-425.

Sales, D., Sae-Lee, D., Matsuya, S. & Ana, I. D. (2003) Short-term fluoride and cations release from polyacid-modified composites in a distilled water, and an acidic lactate buffer. Biomaterials 24, 1687-1696.

Schepers, E., Barbier, L. & Ducheyne, P. (1998) Implant placement enhanced by bioactive glass particles of narrow size range. Int J Oral Maxillofac

Implants 13, 655-665.

Scheyer, E. T., Velasquez-Plata, D., Brunsvold, M. A., Lasho, D. J. & Mellonig, J. T. (2002) A clinical comparison of a bovine-derived xenograft used alone and in combination with enamel matrix derivative for the treatment of periodontal osseous defects in humans. J Periodontol 73, 423-432.

Schopper, C.( 2003) The fluorohydroxyapatite (FHA) FRIOS Algipore is a suitable biomaterial for the reconstruction of severely atrophic human maxillae. Clin Oral Implants Res 14, 743-9

Sculean, A., Stavropoulos, A., Windisch, P., Keglevich, T., Karring, T. & Gera, I. (2004) Healing of human intrabony defects following regenerative periodontal therapy with a bovine-derived xenograft and guided tissue regeneration. Clin Oral Investig 8, 70-74.

Seaborn, C. D. & Nielsen, F. H. (2002) Silicon deprivation decreases collagen formation in wounds and bone, and ornithine transaminase enzyme activity in liver. Biol Trace Elem Res 89, 251-261.

49

Sepulveda, P., Jones, J. R. & Hench, L. L. (2002a) In vitro dissolution of melt-derived 45S5 and sol-gel derived 58S bioactive glasses. J Biomed Mater Res 61, 301-311.

Shchukarev, A. (2006) XPS at solid-aqueous solution interface. Advances in Colloid and Interface Science 122, 149-157.

Sila-Asna, M., Bunyaratvej, A., Maeda, S., Kitaguchi, H. & Bunyaratavej, N. (2007) Osteoblast differentiation and bone formation gene expression in strontium-inducing bone marrow mesenchymal stem cell. Kobe J Med Sci 53, 25-35.

Simao, A. M., Yadav, M. C., Ciancaglini, P. & Millan, J. L. (2010) Proteoliposomes as matrix vesicles' biomimetics to study the initiation of skeletal mineralization. Braz J Med Biol Res 43, 234-241.

Skerry, T. M. (2008) The response of bone to mechanical loading and disuse: fundamental principles and influences on osteoblast/osteocyte homeostasis. Arch Biochem Biophys 473, 117-123.

Soderholm, K. J., Zigan, M., Ragan, M., Fischlschweiger, W. & Bergman, M. (1984) Hydrolytic degradation of dental composites. J Dent Res 63, 1248-1254.

Soucacos, P. N., Johnson, E. O. & Babis, G. (2008) An update on recent advances in bone regeneration. Injury 39 Suppl 2, S1-4.

Stedman, T. L. (2000) Stedman's medical dictionary : illustrated in color. Philadelphia: Lippincott Williams & Wilkins, pp. 2098 s.

TunÇ, E. Ş., ÖZer, L., Sari, Ş. & ÇEtiner, S. (2009) Cytotoxic effects of halogen- and light-emitting diode-cured compomers on human pulp fibroblasts. International Journal of Paediatric Dentistry 19, 55-60.

Vaananen, H. K. & Laitala-Leinonen, T. (2008) Osteoclast lineage and function. Arch Biochem Biophys 473, 132-138.

Wahl, D. A. & Czernuszka, J. T. (2006) Collagen-hydroxyapatite composites for hard tissue repair. Eur Cell Mater 11, 43-56.

Vallet-Regí (2010) Evolution of bioceramics within the field of biomaterials. C. R. Chimie 13.

50

Van der Stok, J., Van Lieshout, E. M., El-Massoudi, Y., Van Kralingen, G. H. & Patka, P. (2011) Bone substitutes in the Netherlands - a systematic literature review. Acta Biomater 7, 739-750.

Van Landuyt, K. L., Nawrot, T., Geebelen, B., De Munck, J., Snauwaert, J., Yoshihara, K., Scheers, H., Godderis, L., Hoet, P. & Van Meerbeek, B. (2011) How much do resin-based dental materials release? A meta-analytical approach. Dental Materials 27, 723-747.

Wennerberg, A. & Albrektsson, T. (2009) Effects of titanium surface topography on bone integration: a systematic review. Clin Oral Implants Res 20 Suppl 4, 172-184.

Williams, D. F. (2009) On the nature of biomaterials. Biomaterials 30, 5897-5909.

Xynos, I. D., Edgar, A. J., Buttery, L. D., Hench, L. L. & Polak, J. M. (2000) Ionic products of bioactive glass dissolution increase proliferation of human osteoblasts and induce insulin-like growth factor II mRNA expression and protein synthesis. Biochem Biophys Res Commun 276, 461-465.

Yazdi, P. M., Schou, S., Jensen, S. S., Stoltze, K., Kenrad, B. & Sewerin, I. (2007) Dentine-bonded resin composite (Retroplast) for root-end filling: a prospective clinical and radiographic study with a mean follow-up period of 8 years. International Endodontic Journal 40, 493-503.

Zimmerli, B., Strub, M., Jeger, F., Stadler, O. & Lussi, A. (2010) Composite materials: composition, properties and clinical applications. A literature review. Schweiz Monatsschr Zahnmed 120, 972-986.

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Papers I-IV