BIOCOMPATIBILITY OF ORTHOPAEDIC IMPLANTS ON BONE FORMING CELLS

ANITAKAPANEN

Department of Anatomy and Cell Biology,and Biocenter Oulu,

University of Oulu

OULU 2002

ANITA KAPANEN

BIOCOMPATIBILITY OF ORTHOPAEDIC IMPLANTS ON BONE FORMING CELLS

Academic Dissertation to be presented with the assent ofthe Faculty of Medicine, University of Oulu, for publicdiscussion in the Auditorium A 101 of the Department ofAnatomy and Cell Biology, on February 22nd, 2002, at 12noon.

OULUN YLIOPISTO, OULU 2002

Copyright © 2002University of Oulu, 2002

Reviewed byProfessor Yrjö KonttinenDoctor Jukka Lausmaa

ISBN 951-42-6606-4 (URL: http://herkules.oulu.fi/isbn9514266064/)

ALSO AVAILABLE IN PRINTED FORMATISBN 951-42-6605-6ISSN 0355-3221 (URL: http://herkules.oulu.fi/issn03553221/)

OULU UNIVERSITY PRESSOULU 2002

Kapanen, Anita, Biocompatibility of orthopaedic implants on bone forming cells Department of Anatomy and Cell Biology and Biocenter Oulu, University of Oulu, P.O.Box 5000,FIN-90014 University of Oulu, Finland 2002Oulu, Finland

Abstract

Reindeer antler was studied for its possible use as a bone implant material. A molecular biologicalstudy showed that antler contains a growth factor promoting bone formation. Ectopic bone formationassay showed that antler is not an equally effective inducer as allogenic material.

Ectopic bone formation assay was optimised for biocompatibility studies of orthopaedic NiTiimplants. Ti-6Al-4V and stainless steel were used as reference materials. The assay showeddifferences in bone mineral densities, with superior qualities in NiTi. The rate of endochondralossification varied between the implants, NiTi ossicles had larger cartilage and bone areas thanossicles of the two other materials.

The cytocompatibility of NiTi was studied with three different methods. Cell viability, celladhesion and TGF-β1 concentration were assessed in ROS-17/2.8 cell cultures. Cells grown on NiTihad better viability than cells grown on pure nickel or stainless steel. Cell attachment on the materialswas studied with paxillin staining of focal contacts. The number of focal contacts was clearly higherin cells grown on NiTi than in cells grown on pure titanium, pure nickel or stainless steel. TGF-β1concentration was measured with ELISA. The results showed that there was only some minorvariation between NiTi, pure titanium and stainless steel. Nickel showed a lower TGF-β1concentration. Taken together, these results suggest that NiTi is well tolerated by ROS-17/2.8 cells.The cytocompatibility of stainless steel is not so good as that of NiTi.

The same tests were used to study the effects of the surface roughness of the implant oncytocompatibility. Three different surface roughness grades were compared in cell cultures on NiTiand titanium alloy discs. Titanium alloy was subjected to two different heat treatments, to comparethe effects of the treatments on cytocompatibility. The studies showed that NiTi had a lesser impacton cell viability and attachment than titanium alloy. Further, rough NiTi was found to be a bettertolerated surface than the others. In this study, heat treatment of titanium alloy at +850° C did notinterfere with cell viability or attachment, as did the +1050° C treatment of the alloy. On the contrary,TGF-β1 concentrations decreased on the +850° C treated alloy and were approximately same on the+1050° C treated alloy and on NiTi.

Keywords: biocompatible materials, osteoblasts, bone morphogenetic proteins, focal adhe-sion, cell death

To the cliffs of the Lake Saimaa

Acknowledgements

This work was carried out at the Department of Anatomy and Cell Biology and BiocenterOulu, University of Oulu, during the years 1996-2001.

I wish to express my deepest gratitude to my supervisor, Professor Juha Tuukkanen,DDS, PhD. His optimism, enthusiastic attitude towards research and everyday life andexcellent sense of humour have been encouragement for me. He should be acknowledgedas the best supervisor one can have.

I am grateful to Professor Yrjö Konttinen, MD, PhD, and Professor Jukka Lausmaa,PhD, for their critical and efficient review of this thesis. I thank Sirkka-Liisa Leinonen,Lic. Phil. for revising the English of the thesis.

I owe my gratitude to Jorma Ryhänen, MD, PhD, for introducing me to the interestingworld of NiTi. You have showed me the way. I thank Anatoli Danilov, PhD, for teachingme the basics of shape memory metals. I appreciate him as a true intellectual, with a verywarm heart. I thank my other co-authors, Joanna Ilvesaro, PhD, and Docent PetriLehenkari, MD, PhD, for fruitful co-operation. Anne Kinnunen is acknowledged for hernever-failing friendship. We have supported each other in this mad world of academicsand shall continue to do so. In addition, to our surprise, we have been co-operating evenoutside the coffee breaks!

I thank my first supervisor, Professor Kalervo Väänänen, MD, PhD, for introducingme into the scientific world. His research group of “Bone Heads” was the best possibleplace to learn research work. I thank Elli Birr, PhD, for the three years we shared withantlers. We certainly had a hard material and hard times, but we also shared thefascinating joy of discovery. I thank Maritta Perälä-Heape, PhD, for mentoring methrough these years.

I thank the whole staff of the Department of Anatomy and Cell Biology. The years Ihave spent at the Anatomy have given me the inspiration to keep going, to move on. I alsowish to thank Minna Vanhala, Marja Paloniemi, Merja Nissilä and Pirkko Peronius fortheir skilful technical assistance.

For the unforgettable moments of womanhood, I want to thank the “girls” of ouroffice: Tuula Kaisto, Hinni Papponen and Marja Nissinen, PhD. Could there be a bettercontraceptive than having to share an office with three mothers?

For maintaining my physical health, I thank the Oriental Dancing Club Yasmine ry.,the Ladies Boxing team of Oulu, and Merja Luukkonen for her excellent massage.

I would like to thank my family. My parents never doubted my prospects as a scientist.I thank them for their support. My two big brothers, Petri and Mika, gave me enough ofhard times at home to make me manage in the outside world. I guess, no matter howmasochistic it might sound, this should be gratefully acknowledged. Finally, I want toexpress my deepest gratitude to my Rauski. Without his never-failing love and tenderness,this work would have taken a whole lot longer to accomplish. I am lucky to have a personlike him by my side.

This research was financially supported by the Ministry of Agriculture and Forestry,the National Technology Agency (TEKES), Research and Science Foundation of Farmosand the Pohjois-Pohjanmaa Fund of the Finnish Cultural Foundation.

Oulu, January 2002 Anita Kapanen

Abbreviations

AFM Atomic force microscopyAISI American Iron and Steel InstituteALP Alkaline phosphataseAMV Avian Myeloblastosis virusAO/ASIF Arbeitsgemeinschaft für Osteosynthesefrage/Association for the

Study of Internal FixationAs Austenite start temperatureASTM American Society for Testing and MaterialsAuCd Gold-cadmium alloyBlastN Basic local alignment tools, nucleotidesBlastP Basic local alignment tools, proteinsBlastX Basic local alignment tools, both nucleotides and proteinsBMD Bone mineral densityBMP Bone morphogenetic proteinBMPR Bone morphogenetic protein receptorBSP Bone sialoproteinCBFA-1 Core-binding factor-alpha 1CDMP Cartilage-derived morphogenetic proteincDNA Complementary deoxyribonucleic acidCo-Cr-Mo Cobalt-chromium-molybdenum alloyCu-Al-Ni Copper-aluminium-nickel alloyDNA Deoxyribonucleic acidDTT dithiotreitoldUTP deoxyurasiltriphosphateELISA Enzyme-linked immunosorbent assayGDF Growth and differentiation factorHCl Hydrochloride acidICAM Intercellular adhesion moleculeIL InterleukineInTl Indium-tellurium alloyMd Highest temperature to strain-induced martensite

Mf Martensite finish temperatureMg MagnesiumMs Martensite start temperatureMT Martensite transformationMTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideNi NickelNiO Nickel oxideNi3S2 Nickel sulphideNiTi Nickel-titanium alloyOP OsteopontinOP-1 Osteogenic protein-1PBS Phosphate-buffered salinePDGF Platelet-derived growth factorPFA ParaformaldehydepQCT Peripheral quantitative computed tomography5’/3’ RACE Rapid amplification of cDNA sequence endsRGD Arginine-glycine-aspartic acidRNA Ribonucleic acidRT-PCR Reverse transcriptase polymerase chain reactionROS-17/2.8 Rat osteosarcoma cell lineSMA Shape memory alloySME Shape memory effectStst Stainless steel alloyTdT Terminal deoxynucleotidyl transferaseTGF-β1 Transforming growth factor-β1Ti TitaniumTi-6Al-4V Titanium-aluminum(6%)-vanadium(4%) alloyTiO2 Titanium oxideTi-6Al-2.2Mo-1.3Cr Titanium-aluminum(6%)-molebdenum(2.2%)-chromium(1.3%)TNF-β Tumor necrosis factor-βTTR Transition temperature rangeTUNEL Terminal deoxynucleotidyl transferase-mediated dUTP nick end la-

belingUV UltravioletVgr Vegetal related

Definitions

Allograft Material taken for grafting from another individual ofthe same species.

Austenite The high-temperature (parent) phase of some metals, e.gNiTi.

Autograft Material taken from the same individual.Biocompatibility A general term used to describe the suitability of a mate-

rial for exposure to the body or bodily fluids. The specif-ic meaning is dependent upon the particular applicationor circumstances.

Biomaterial A material intended to come into contact with biologicalsystems.

Cytocompatibility The ability of a material to perform an appropriate cellu-lar response.

Graft A material to be transplanted into the bodyHysteresis In case of SMA, the difference of temperature at which

the material is 50% transformed to austenite upon un-loading and 50% transformed to martensite upon stress.

Implant A medical device made of one or more biomaterials thatis placed within the body.

Martensite The low-temperature phase of some metals, e.g. NiTi.Martensite transformation A lattice transformation involving shearing deformation

and resulting from cooperative atomic movementOsteoconduction Ability to guide bone formation on a material surface in

a bony environment.Osteoinduction Ability to induce bone formation in non-osseous tissue.Shape memory alloy Material with an ability to return to some previously de-

fined shape or size when subjected to an appropriatestress procedure.

Shape memory effect An alloy whose fixed shape has been stored, which, afterdeformation by stress followed by stress release, revertsto its original shape.

Superelasticity (pseudoelasticity) The ability of a material to fully recover its initial shapeupon removal of the load under isothermal conditions.

Transition temperature Temperature at which changes of material phases occur.Xenograft Material taken from an individual of another species.

List of original publications

This thesis is based on the following articles, which are referred to in the text by theirRoman numerals.

I Kapanen A, Ryhänen J, Birr E, Väänänen K & Tuukkanen J (2002) Bone morphoge-netic protein 3b expressing reindeer antler. J Biomed Mat Res 59:78-83.

II Kapanen A, Ryhänen J, Danilov A & Tuukkanen J (2001) Effect of nickel–titaniumshape memory metal alloy on bone formation. Biomaterials 22:2475-2480.

III Kapanen A, Ilvesaro J, Danilov A, Ryhänen J, Lehenkari P & Tuukkanen J (2002)Behaviour of Nitinol in osteoblast-like ROS-17 cell cultures. Biomaterials 23:645-650.

IV Kapanen A, Danilov A, Lehenkari P, Ryhänen J & Tuukkanen J. Effect of metalalloy surface roughness on the viability of ROS-17/2.8 osteoblastic cells. Submitted

V Kapanen A, Kinnunen A, Ryhänen J & Tuukkanen J. TGF-β1 secretion of ROS-17/2.8 cultures on NiTi bone implant. Biomaterials, in press.

Contents

Abstract Acknowledgements Abbreviations Definitions List of original publications 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 Review of the literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.1 Bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182.1.1 Structure and function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182.1.2 Bone-forming cells: osteoblasts, osteocytes, lining cells . . . . . . . . . . . . . . 192.1.3 Remodelling cycle of bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212.1.4 Bone morphogenetic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2.2 Biomaterials in orthopaedics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242.2.1 Shape memory metal NiTi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242.2.2 Cytotoxicity of nickel and titanium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

2.2.2.1 Biocompatibility studies of NiTi in vitro . . . . . . . . . . . . . . . . . . . . 272.2.2.2 Biocompatibility studies of NiTi in vivo . . . . . . . . . . . . . . . . . . . . 28

2.2.3 Surface of implant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 Aims of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

4.1 Decalcified bone materials (I, II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334.2 Test materials (II-V) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334.3 Molecular biology methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

4.3.1 Isolation and characterisation of reindeer BMP-3b cDNA (I) . . . . . . . . . . 344.3.2 In situ hybridisation (I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344.3.3 Apoptosis detection (III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

4.3.3.1 DNA laddering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354.3.3.2 Terminal deoxynucleotidyl tranferase (TdT)-mediated dUTP

nick end labeling (TUNEL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354.4 Cytotoxicity test (III, IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364.5 Enzyme-linked immunosorbent assay (ELISA) (V) . . . . . . . . . . . . . . . . . . . . . . 364.6 Cell line (III-V) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364.7 Animals (I,II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

4.8 Ectopic bone formation assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374.9 Methods of analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

4.9.1 Peripheral quantitative computed tomography (pQCT) (I, II) . . . . . . . . . . 374.9.2 Light microscopy (I, II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384.9.3 Bone histomorphometry (I, II) and digital image analysis (I-IV) . . . . . . . 384.9.4 Confocal laser scanning microscopy (III, IV) . . . . . . . . . . . . . . . . . . . . . . 384.9.5 Atomic force microscopy (AFM) (IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394.9.6 Statistical analysis (I-V) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

5 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405.1 Bone induction capacity of decalcified reindeer antler matrix (I) . . . . . . . . . . . . 40

5.1.1 Isolation and characterisation of BMP-3b cDNA (I) . . . . . . . . . . . . . . . . . 405.1.2 Sequence analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405.1.3 In situ hybridisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415.1.4 Ectopic bone formation assay of antler matrix . . . . . . . . . . . . . . . . . . . . . . 41

5.2 Biocompatibility studies of NiTi (II-V) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425.2.1 Ectopic bone formation assay (II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425.2.2 Surface roughness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435.2.3 Cell viability (III, IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435.2.4 Apoptosis of cells (III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445.2.5 Cell attachment (III, IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445.2.6 Detection of TGF-b1 and IL-6 cytokines (V) . . . . . . . . . . . . . . . . . . . . . . 46

6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476.1 Bone induction capacity of decalcified reindeer antler matrix (I) . . . . . . . . . . . . 47

6.1.1 Isolation and characterisation of BMP-3b cDNA . . . . . . . . . . . . . . . . . . . 476.1.2 Sequence analysis and in situ hybridization . . . . . . . . . . . . . . . . . . . . . . . . 476.1.3 Ectopic bone formation assay of antler matrix . . . . . . . . . . . . . . . . . . . . . . 48

6.2 Biocompatibility studies of NiTi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496.2.1 Ectopic bone formation assay (II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496.2.2 Cell viability (III, IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496.2.3 Apoptosis of cells (III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506.2.4 Cell attachment (III, IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516.2.5 Detection of TGF-b1 and IL-6 cytokines (V) . . . . . . . . . . . . . . . . . . . . . . 52

7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

1 Introduction

Bone fracture induces a chain of cellular and molecular events at the fractured site.Surgery is often needed for proper bone healing. The biomaterials used in orthopaedicsvary a lot as to their source and nature, but they are expected to share some commonproperties. Biomaterials, including orthopaedic implants, should not cause host responses,such as tissue necrosis. Osteolysis, bone resorption and the formation of a thick fibroticcapsule indicate poor biocompatibility. The implant should have resistance againstmechanical load, which is one of the basic properties of healthy bone. It wouldsometimes also be beneficial if the implant could stimulate bone formation.

Some metals are suitable for load-bearing implants because of their mechanicalstrength and biocompatibility. An increase of implant wear, however, sometimesincreases the surface area and the concentration of metal ions. It is therefore importantthat the mechanical properties of the implants and their surface are able to minimize ionleakage. Implants that stimulate bone formation include hydroxyapatite-based materialsor auto-, allo- and xenografts of bone. Allo- and xenografts may lead to problem due toimmunologic host reactions. Depending on the case, a combination of load-bearing metaland bone formation-stimulating material in the same implant might be better than a metalor bone-inductive implant alone.

The experimental part of this project focused on the methods that can be used to studythe bio- and cytocompatibility of orthopaedic biomaterials. We here concentrated on NiTi,which is a shape memory metal alloy. In addition, the possibility to use reindeer antler toinduce bone formation was studied. The bone induction properties of antler were studiedusing molecular biological methods and ectopic bone formation assay. We optimized theectopic bone formation assay to study the biocompatibility of NiTi. Further, cell culturestudies were done to clarify the effects of NiTi on cell viability, attachment and cytokinerelease. The surface roughness effects on cells were also examined. Pure elements of thealloy, i.e. titanium and nickel, and some commonly used orthopaedic implant materialswere used for comparison.

2 Review of the literature

2.1 Bone

2.1.1 Structure and function

Bone constitutes most of the skeleton of higher vertebrates. It provides mechanicalstrength, shelter for internal organs and a place for blood formation and plays animportant role in the calcium homeostasis of the body. Bone consists of intercellularmatrix and bone-forming cells, i.e. osteoblasts, osteocytes, lining cells and bone-resorbing cells, i.e. osteoclasts. The intercellular matrix is composed mostly of collagentype I as the major organic compound and inorganic components, such as calciumphosphate. Inorganic salts account for two thirds of the weight of bone and areresponsible for the hardness of osseous tissue.

There are two different types of bone: cancellous and cortical. Cancellous boneconsists of irregular bars, or trabeculae, that branch and form a mesh, which is filled withbone marrow. Cancellous bone has very active metabolic functions due to its proximity toand extensive interphase with bone marrow. Cortical bone is compact and has protectiveand mechanical functions. In long bones, cortical bone covers the cancellous bone like atube.

Bones may develop via two mechanisms: intramembranous and endochondralossification. Both types are active in embryonic development and in adult bonerenovation. Intramembranous ossification occurs in flat bones of the skull. In thedeveloping skull, mesenchymal tissue is strongly vascularised and some mesenchymalcells differentiate directly into osteoblasts. Mineralisation is initiated in the matrixvesicles that bud away from the plasma membrane of the osteoblast, similarly to cartilagemineralization (Hohling et al. 1978). Matrix vesicles are extracellular organelles, whichcontain proteinases, peptidases, and alkaline phosphatase (ALP) (Hirschman et al. 1983,Dean et al. 1994, Wuthier et al. 1985). Further, hydroxyapatite crystals are first seen onvesicle membrane, potentially serving as nuclei for subsequent bulk calcification of theextracellular matrix (Anderson 1969). Endochondral ossification typically occurs in long

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bones. Mesenchymal cells first differentiate into chondroblasts to form a cartilaginousmodel, which is subsequently mineralised. Matrix-resorbing osteoclasts, which havedifferentiated from their heamotopoetic precursors, remove the mineralised cartilage,after which the mesenchymal osteoprogenitor cells differentiate into osteoblasts. Theyproduce new bone, while the remnants of calcified cartilage form a supportingframework. Mineralization begins by the synthesis of components of collagen ornoncollagenous proteins (Marks & Hermey 1996). Transcription factor CBFA-1 has a keyrole in bone formation (Ducy et al. 1997). CBFA-1 knock-out mice (mice without theCbfa-1 gene) had no bone formation, because both endochondral and intramembranousossification were eliminated (Komori et al. 1997, Otto et al. 1997).

Antlers, the result of a peculiar form of endochondral bone formation, are bony cranialorgans unique to the Cervidae family (Chapman D.I. 1975). Antlers differ from Bovidaehorns in that they are cast every year and that antler growth takes place at the tip (Goss1983b). Regenerating antler is covered by skin with thick, fine hair. Therefore, growingantler is called “velvet antler”. The antler grows by a process of modified endochondralossification; that is, bone formation takes place in a preformed frame of cartilage, whichis highly vascularised (Banks 1974). There is a zone of fibroblasts at the very tip of theantler, followed by layers of cartilage, calcified cartilage and ultimately bone, forming agradient of progressive differentiation from the zone of active cell division at the tip tomineralised bone at the antler root (Suttie & Fennessy 1990). Superficial arterioles supplythe tip, and venous drainage takes place through the antler core (Suttie et al. 1985).Antlers are innervated mainly by sensory nerves (Gray et al. 1992) Antlers form aninteresting model of adult regenerating mineralised tissue (Price et al. 1994;Price et al.1996). Bone remodelling has been shown to continue until the time of antler casting (Rolf& Enderle 1999). Both reindeer and another Cervidae, moose (Alces alces), have beenused for the production of BMP implantation material. The BMP fraction produced hasbeen shown to be highly active (Viljanen et al. 1996). Three specific BMPs have beensequenced from antler cDNA: BMP-4, -2 and BMP-3b (Feng et al. 1995). In addition,antlers have been used to monitor environmental pollution by bone-seeking pollutants,such as fluoride (Kierdorf & Kierdorf 2001).

2.1.2 Bone-forming cells: osteoblasts, osteocytes, lining cells

The cellular origin of bone was recognised in the early 19th century, and the term“osteoblast” was first used by Gegenbaur 1864 to refer to the “granular corpuscles foundin all developing bone as the active agents of osseous growth” (Gegenbaur 1864).

The bone-forming cell, i.e. osteoblast, is of mesenchymal origin and its main functionis to produce new bone matrix, osteoid, and to mineralise it. During differentiation, theosteoblast goes through morphological and functional changes. The preosteoblast is flatand expresses transforming growth factor-β (TGF-β), which induces osteoblast cellproliferation (Noda & Camilliere 1989) (figure 1). In addition, osteopontin (OP) isexpressed, even though its expression peaks later. The proliferating osteoblast expressesproteins needed in cell division, such as histones, c-fos and c-myc, but early also the mainprotein of matrix, collagen type I. Mature osteoblasts are more cuboidal and express ALP,

20

which is an important enzyme for osteoid mineralisation. At the next stage, the cellbecomes fully differentiated and cuboidal in shape. A differentiated osteoblast expressesosteopontin and a closely structurally related protein known as bone sialoprotein (BSP).Finally, during the mineralisation phase, the cell produces osteocalcin, a majornoncollagenous protein in bone (Stein et al. 1996).

Approximately 50-70 % (Jilka et al. 1998) of osteoblasts die trough programmed celldeath, apoptosis, while the rest are embedded in the matrix or form a layer of resting cellson the bone surface. The embedded cells are called osteocytes, and they are responsiblefor the maintenance of bone. Osteocytes constitute a functional syncytium, whichcommunicates through dendritic processes and gap junctions (Jones et al. 1993). Thissyncytium also includes bone-lining cells or osteoblasts, depending on whether the bonein question is resting or remodelling (Palumbo et al. 1990). Osteocytes may communicatewith bone-resorbing osteoclasts. Both osteoclasts and osteocytes, when located in closecontact with each other, contain antigen CD44, which is macrophage lineage specific, andosteopontin, which is an osteoblastic protein (Yamazaki et al. 1997).

The osteoblasts that constitute a resting layer instead of dying through apoptosis arecalled lining cells. A lining cell is an inactive, postproliferative cell covering a bonesurface that is undergoing neither bone formation nor resorption. Studies have shown thatthese cells can be reactivated into bone-producing osteoblasts (Chow et al. 1998). Theyare thought to be responsible for the activation of bone remodelling by producingcytokines and other signals, which activate osteoclasts. The osteocytic syncytiumproduces and transmits signals proportional to mechanical loading (Martin 2000a).Marotti et al. 1992 suggested that osteocytes can send an inhibitory signal to osteoblaststhat reduces their rate of bone formation (Marotti et al. 1992). This hypothesis has beendeveloped further by Martin 2000, who has described an osteocytic inhibitory signalproduced in response to mechanical loading able to inhibit bone lining cells fromactivating remodelling (Martin 2000a).

Osteoblast differentiation and activity is regulated by many hormonal and autocrine/paracrine factors. An osteoblast has several membrane receptors for these factors, andbinding of these factors to their receptors activates signal transduction pathways thatfinally lead to nuclear responses. These hormonal factors include estrogen andparathyroid hormone. Local factors, which affect directly the same cell or neighbouringcells, include bone morphogenetic proteins (BMPs) and their antagonists, noggin andchordin.

Fig. 1. A schematic presentation of osteoblast protein expression in different stages of the cell’slife (Stein et al. 1996).

Preosteoblast Proliferating osteoblast

Maturating osteoblast

Differentiating osteoblast

Mineralization

TGF-β, osteopontin

Histones, collagen type I

Alkaline phosphatase

Osteopontin, bone sialo prot.

Osteocalcin

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2.1.3 Remodelling cycle of bone

Bone tissue is under constant reconstruction. Approximately 30% of bone mass isremodelled in a year. This is necessary for normal skeletal maintenance. Bone matrix isproduced and mineralised by osteoblasts, as described in the previous chapter. Boneresorption is done by bone-resorbing cells, osteoclasts. Osteoclasts originate from thehematopoetic-macrophage lineage. Their mononuclear precursors use vascular routes toenter skeletal sites, where they fuse to become active, multinucleated osteoclasts. By amechanism still unknown, osteoclasts are guided to appropriate sites to be resorbed.There are theories postulating that osteocytes, osteoblasts and bone-lining cells regulatethe ionic flow between the syncytium and the extracellular fluid of bone. Thiscommunication network seems to influence two bone cell activities: strain-relatedadaptive remodelling and mineral exchange (Rubinacci et al. 1998).

During bone resorption, osteoclasts go through several steps, including attachment tothe bone surface, polarisation of the cell surface into three distinctive membranecompartments, formation of a sealing zone, resorption, final detachment and eventual celldeath (Väänänen 1996). Resorption of bone leads to a release of the growth factors buriedin the bone matrix, such as TGF-β, BMPs and other factors that activate and recruitosteoblasts to form new bone at the resorption site. Osteoblasts, in turn, produce growthfactors, such as BMPs and PDGF, which are embedded in the newly synthesised bonematrix, and cytokines, which modulate osteoclast activity, including IL-11, IL-6, IL-8 andTNF-β (Bilbe et al. 1996).

Normal fracture healing produces bone with properties similar to preexisting tissue.Many of the cellular and biochemical events that occur during fracture healing areidentical to those taking place in the growth plate during development, with the exceptionthat these events occur on a temporal rather than a spatial scale. Fracture healing may beviewed as consisting of four distinct responses occurring in different tissue compartments.They take place in bone marrow, cortex, periosteum and external soft tissue. In bonemarrow, there is a rapid transformation of endothelial cells into osteoblasts (Brighton &Hunt 1991). The cortex may either directly re-establish itself (primary cortical healing) orform a callus, which is associated with responses in the periosteum and external tissue(secondary fracture healing). The majority of fractures heal by the mechanism ofsecondary fracture healing. In the periosteum, osteoprogenitor cells and undifferentiatedmesenchymal cells contribute to the healing process by intramembranous andendochondral ossification. Intramembranous ossification occurs farther from the site offracture, resulting in the formation of hard callus. Intramembranous bone is formed byosteoprogenitor cells of periosteum. Adjacent to the fracture site, bone is formed byendochondral ossification. Secondary fracture healing starts with the formation of ahematoma and inflammation. Inflammatory cells secreting cytokines, such as IL-1 andIL-6, may have regulatory roles in the early events of fracture healing (Einhorn et al.1995). At the second stage, vascular formation proceeds and cartilage begins to form,followed by cartilage calcification and removal of calcified cartilage followed by boneformation. Finally, bone remodelling begins to take place.

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2.1.4 Bone morphogenetic proteins

Bone morphogenetic proteins (BMPs) are a large family of secreted polypeptides withcommon structural features and diverse effects on tissue development (table 1). Thealignment of amino acid sequences of BMPs indicates significant amino acid sequenceidentity in the carboxy-terminal region of the proteins. BMPs are synthesised within cellsas precursor forms. They have a hydrophobic leader sequence, a long propeptide portionand a carboxy-terminal mature peptide domain. In the mature region, BMPs have sevenconserved cysteines (figure 2). These cysteine residues are important for the three-dimensional structure of the protein. Cysteine residues 1-3 and 4,6-7 form disulfide bondsin the BMP monomer, but the fifth cysteine residue bonds with the complementaryresidue in the other BMP monomer to produce a dimer (Griffith et al. 1996). Afterproteolytic cleavage of the leader sequence and the propeptide, the mature regions formdimers intracellularly (Hazama et al. 1995). Their role in bone remodelling and fracturehealing is discussed briefly.

Fig. 2. Structure of BMP proteins deduced from cDNA clones. A generic BMP molecule isshown schematically with its secretory leader sequence, pro-peptide region, and carboxy-terminal mature region. BMPs contain potential N-linked glycosylation sites (Y) in both thepropeptide and the mature regions. Seven conserved cysteine residues are marked as C in themature region. RXXR marks the conserved sequence at the proteolytic cleavage site.

Among the various factors that could be used to facilitate fracture healing, theexpression of bone morphogenetic proteins is thought to be important. During fracturerepair, BMPs are needed for the initiation of cell differentiation. The further thedifferentiation of chondroblasts and osteoblasts advances, the less they express BMP-2, -4, and -7 and their receptors BMPR-IA, -IB and II in these cells (Ishidou et al. 1995).Only BMP-6 has been shown to be expressed in a more differentiated cell, namelyhypertrophic chondrocyte (Grimsrud et al. 1999). There are several preclinical animalstudies showing the potential of BMPs, mostly BMP-2 and -7, to enhance fracture healing(Riley et al. 1996). Recently, recombinant human BMP-7 has been introduced into tibialnonunions of human patients with good results (Friedlaender et al. 2001).

C C C C C CRXXRPropeptide Mature regionLeader Y Y Y

15-25 aa

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Table 1. Bone morphogenetic protein superfamily.

BMP Major expression tissue

Other names References Mammalian cDNA sequenced

BMP-2 Cartilage, bone BMP-2a Wozney et al. 1988,Sampath et al. 1990Feng et al. 1994, 1997

Bovine, human, mouse, rat, reindeer

BMP-3 Cartilage, bone, ovary Osteogenin Wozney et al. 1988, Takao et al. 1996

Bovine, human, rat

BMP-3b Muscle, bone, brain, lung

GDF-10 Cunningham et al. 1995Hino et al. 1996, Takao et al. 1996 Kapanen et al. 2000

Human, rat, mouse, reindeer

BMP-4 Cartilage, bone, prostate

BMP-2b Wozney et al. 1988, Feng et al. 1995

Human, mouse, reindeer

BMP-5 Bone Celeste et al.1990 Bovine, human

BMP-6 Cartilage Vgr-1 Celeste et al.1990Gitelman et al.1994

Bovine, human, mouse, rat

BMP-7 Bone, kidney OP-1 Celeste et al.1990Simon et al. 1999

Bovine, human, mouse, rat

BMP-8a,b Testis, placenta OP-2, OP-3 Özkaynak et al. 1990, Zhao et al. 1996

Human, mouse

BMP-9 Liver GDF-2 Song et al. 1995 Human

BMP-10 Bone, heart Celeste et al. 1995, Neuhaus et al. 1995

Human, mouse

BMP-11 Neurogenesis Celeste et al. 1995 Human, mouse

BMP-12 Cartilage GDF-7, CDMB-3 Strom et al. 1994,Celeste et al. 1995

Human, mouse

BMP-13 Cartilage GDF-6, CDMB-2 Strom et al. 1994, Dube & Celeste 1995, Chang et al. 1994

Human, mouse, bovine

BMP-14 Cartilage GDF-5, CDMB-1 Strom et al. 1994, Chang et al. 1994

Bovine, mouse, human

BMP-15 Oocytes GDF-9 McPherron & Lee 1993, Dube et al. 1998

Human, mouse

GDF = Growth and differentiation factor, OP = Osteogenic protein, Vgr = Vegetal-related, CDMP = Cartilage-derived morphogenetic protein

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2.2 Biomaterials in orthopaedics

Biomaterials are inorganic or organic natural or synthetic materials placed in the body.Biomaterials are expected to be biocompatibile, i.e. they should not cause inflammationor rejection. Orthopaedic biomaterials can be implanted into or near a bone fracture tofacilitate healing or to compensate for a lack or loss of bone tissue. The materials used inorthopaedic surgery include ceramics, polymers, metals, such as stainless steel, cobalt-chromium and titanium and the shape memory alloy NiTi, and resorbable materials, suchas bioglass, various modifications of hydroxyapatite and bone grafts.

An implant may have bioactive effects on ossification. It may mediate recruitment ofmesenchymal cells by growth factors derived from the implant, for example, a bone graft.This is called osteoinduction. In addition, the implant may provide three-dimensionalframes for the ingrowth of capillaries and osteoprogenitor cells. In this case, the implanthas osteoconductive properties. However, metal alloy implants often give support to bonetissue without any active role in bone formation. The properties of NiTi are herediscussed in more detail.

2.2.1 Shape memory metal NiTi

The term “shape memory alloy” (SMA) is used about a group of metal alloys that havethe ability to return to some previously defined shape or size when subjected to anappropriate stress procedure. The first records of SMA were published about gold-cadmium (AuCd) alloy in 1932 (Chang & Read 1951). In 1962, Buehler and co-workersdiscovered the shape memory effect in equiatomic nickel-titanium alloy (NiTi)(Buehler& Wang 1968). NiTi is also referred to as Nitinol, which is an abbreviation from thewords: nickel-titanium Naval Ordnance Laboratory.

Typical properties of SMA are due to a phenomenon known as phase transformation.In phase transformation, atoms re-organise. Re-organisation where only smalldisplacements of atoms occur is called martensitic transformation (MT). Thistransformation does not change the chemical composition, but results in a new crystallattice. Thermal cycling, external strain or stress can be used to induce this kind oftransformation.

Four temperatures characterise temperature-induced transformation. Ms (martensitestart temperature) and Mf (martensite finish temperature) are the temperatures duringcooling or loading at which transformation from the parent phase into martensite startsand ends. (Fig. 3) The reaction induced by heating or unloading that results in reversemartensitic transformation is characterised by the temperatures As (austenite starttemperature) and Af (austenite finish temperature). There is a slight difference betweenthe temperature range of forward and reverse transformations due to the energeticallydissimilar transformation pathways. The temperature difference between the phasetransformations upon heating and cooling is called hysteresis. In NiTi alloys, it isgenerally measured as the difference between the temperatures at which 50% of thematerial is transformed to austenite and 50% to martensite. Finally, the temperature or

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load beyond which the SMA behaves like normal metal is called the martensiticdeformation temperature Md (Humbeeck et al. 1998)

Fig. 3. Schematic thermal transformation hysteresis loop. T = temperature, %M = martensitepercentage, Ms = martensite start, Mf = martensite finish, Md = martensite deformation, As=austenite start, Af = austenite finish, H = hysteresis.

The shape memory effect (SME) is defined as the capacity of a material to recover agiven strain upon stress release and/or heating. At least three distinctly observable effectsare compatible with this general definition (Humbeeck et al. 1998).1. The shape memory effect per se takes place when the material is deformed at a

temperature below Mf and then heated above Af. One characteristic of this effect isthe “strain limit”, beyond which strain recovery is incomplete and the material nolonger exhibits any memory effect.

2. Two-way (reversible) shape memory is a phenomenon where a martensitic shape isobtained spontaneously during cooling without any external forces. During heating,the original austenite phase is recovered. The characteristic feature of this effect is alack of hysteresis during temperature cycling. Typically, the magnitude of the two-way shape memory effect is of the order of 1%, and it therefore lacks any realpractical interest.

3. Superelasticity is an isothermal phenomenon involving storage of potential energy.Material deformed beyond its apparent yield point fully recovers its initial shape onremoval of the load. Significant stress-strain hysteresis is typical of this effect.

In near-equiatomic NiTi alloys, the shape memory effect results from a thermoelasticmartensitic reaction that occurs as the alloy is cooled through a critical transitiontemperature range (TTR) (Abujudom et al. 1990). As the alloy is cooled through theTTR, the highly symmetric cubic (B2-type) austenitic (β) phase transforms throughsmall-scale coordinated atom displacements in a martensitic phase of low crystalsymmetry (B19’).

Factors such as nickel content, aging, thermo-mechanical treatment and addition ofalloying elements are important factors in the control of memory behaviour. From thepoint of view of practical applications, NiTi can exist in three different, temperature-dependent forms: martensite, superelastic and austenite. The martensitic form is ductile,soft and easily deformed. Superelastic NiTi is highly elastic. Lastly, austenitic NiTi is

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hard, resembling titanium. Commercial NiTi alloys are well used because of theirproperties. NiTi alloys have large reversible deformations (up to 8 %) at nearly constantstress levels, tend to be thermally stable and have high ductility and corrosion resistance.The disadvantages of NiTi include the high price of the material, which is partly due itsproblematic production, where a vacuum or an inert atmosphere is needed due to thereactivity of titanium, and the alloy has fewer transformation temperatures than, forexample, copper-based alloys.

Despite these exceptional physical, chemical and mechanical properties, orthopaedicapplication of NiTi has been difficult due to the lack of knowledge of the biocompatibilityof NiTi on bone.

2.2.2 Cytotoxicity of nickel and titanium

Release of metallic elements from almost all types of alloys has been documented (Brune1986). The cellular effects of metallic elements do not seem to depend on the type of thecell, which points towards a common mechanism via which cells are affected by metalliccompounds (Yamamoto et al. 1998). The cytotoxicity of nickel and titanium has beenwidely studied, especially in the case of nickel, which is a toxic agent and allergen.

Titanium is considered to be a well-tolerated and nearly inert material (Albrektsson etal. 1981). In an optimal situation, titanium is capable of osteointegration with bone(Branemark et al. 1969). Further, titanium is able to form a calcium phosphate-rich layeron its surface (Hanawa & Ota 1991) Moreover, titanium forms a stable titanium oxidelayer on its surface (Kasemo & Lausmaa 1991). This feature is responsible for the goodbiocompatibility of titanium. Particles derived from titanium implants consist mostly ofinsoluble titanium oxides or suboxides. They arise from the passivation layer of theimplant. If the implant layer is damaged for some reason, the layer is immediatelyreoxidised. This property protects the alloy and prevents the formation of chemicalcompounds other than oxides (Hildebrand & Hornez 1998) Titanium is considered one ofthe best-accepted metals in vitro and in vivo (Doran et al. 1998).

However, in vitro Ti4+ ions inhibit osteoclastic activity and reduce osteoblastic proteinsynthesis (Thompson & Puleo 1996). In a study using the human osteoblastic cell lineMG-63, which can be defined as proliferating osteoblasts, titanium was shown to induceIL-6 production (Shida et al. 2000) and, therefore, activate osteoclastogenesis (Kuriharaet al. 1990). In addition, there are reports of contact dermatitis in response to titanium(Layor et al. 1991).

Nickel is well-known for its toxicity and its propensity to cause allergies. It is oftenneglected, however, that nickel is an essential element for the human body. The dietaryexposure to nickel is 160-600 mg/day, with most of nickel being eliminated in the feces,urine and sweat. Nickel is one of the structural components of the metalloproteins.

Nickel can enter the cell via various routes. Ni2+ ions may enter the cell utilising thedivalent cation receptor (Quarles et al. 1997) or via the Mg2+ channel, which are bothsituated in plasma membrane. Nickel-containing particles can be phagocytosed by thecell. Phagocytosis of nickel-containing compounds is enhanced by their crystallinenature, negative surface energy, appropriate particle size (2-4 µm) and low solubility

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(Sunderman, Jr. et al. 1987). Ni3S2 and NiO with low in vivo solubility are thought todepend largely upon this pathway (Dunnick et al. 1995). There are two intracellularpathways for nickel. Soluble nickel, such as Ni2+ ions, which enters through receptors orion channels, binds to cytoplasmic proteins and does not accumulate in the cell nucleus atconcentrations high enough to cause genetic consequences (Abbracchio et al. 1982a).Such soluble Ni2+ ions are rapidly cleared from the body (Oller et al. 1997). In contrast,the insoluble nickel particles containing phagocytotic vesicles fuse with lysosomes. Thisis followed by a decrease of phagocytic intravesicular pH, which releases Ni2+ ions fromnickel-containing carrier molecules. This contributes to the formation of oxygen radicals,DNA damage and thereby inactivation of tumour suppressor genes (Klein et al. 1991a).Nickel is harmful in bone tissue cultures, but less so than cobalt or vanadium (Yamamotoet al. 1998).

Ni2+ ions increase the proliferation of rabbit bone marrow-derived osteoblasts, butinhibit their maturation and ALP activity and retard mineralisation (Morais et al. 1998).On the contrary, nickel chloride decreased the proliferation of both chondrocytes andfibroblasts (Grant et al. 1994). In addition, nickel inhibits enzymes important for theprotection of tissues against oxidative agents (Rodriquez et al. 1990).

2.2.2.1 Biocompatibility studies of NiTi in vitro

Cytotoxicity of and cellular tolerance for NiTi have been studied in various cell culturemodels. Human monocytes and microvascular endothelial cells were exposed to purenickel, titanium, stainless steel and NiTi. The secretion of Il-1β from monocytes andICAM-1 expression on endothelial cells were measured. NiTi significantly enhanced IL-1β secretion by monocytes. This secretion was sufficient to induce ICAM-1 expressionon endothelial cells. Somewhat surprisingly, it was found that stainless steel actuallyreleased similar quantities of nickel without activating monocytes. Therefore, the releaseof nickel is not the only reason for the results (Wataha et al. 1999). NiTi was shown torelease higher concentrations of Ni2+ in human fibroblast and osteoblast cultures, but thisdid not seem to have any effect on cell growth (Ryhänen et al. 1997).

In vitro studies of minimal essential medium extract cytotoxicity, guinea-pigsensitisation, genotoxicity and salmonella reverse mutation test indicated that NiTibehaves similarly to stainless steel. NiTi did not induce any toxic, allergic or geneticeffects (Wever et al. 1997).

Genotoxicity of NiTi was also studied in a peripheral blood lymphocyte model with insitu end labeling and electron microscopy. NiTi caused significantly less single-strandbreaks on interphase chromatin than pure nickel. Additionally, stainless steel had almostthe same effect as nickel. A study of metal ion release in semiphysiological mediumrevealed very low concentrations of nickel and titanium that were released from NiTi.The authors concluded that NiTi had no genotoxic effects (Assad et al. 1999).

Depending on the corrosion resistance test used, the results of NiTi corrosion havediffered. Rondelli et al. showed that NiTi alloys have different corrosion resistance valueswhen measured in artificial saliva or isotonic saline or with the ASTM F764 standard test(Rondelli & Vicentini 1999). The results on the corrosion resistance of Niti and

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biocompatibility have been inconsistent. In a study done in simulated physiologicalsolutions, the anodic polarisation behaviours of NiTi and Ti-6Al-4V were compared. NiTiwas considered to be biocompatible (Speck & Fraker 1980). However, a surface corrosionstudy of NiTi and stainless steel under clinical conditions showed low corrosionresistance and, therefore, low biocompatibility for NiTi (Edie et al. 1981). This negativeeffect was also seen in a comparison of four orthodontic wires in a chloride-inducedcorrosion test (Sarkar et al. 1983).

It is possible that the concentration of nickel affecting cells is higher in vitro than intissue environments in vivo. Surface characterisation and comparison of NiTi to Ti-6Al-4V and stainless steel revealed a TiO2-based layer on both NiTi and Ti-6Al-4V with a lowconcentration of nickel. Furthermore, a calcium-phosphate layer formed around NiTi,similar to that formed around titanium alloys (Wever et al. 1998). Similar findings werealready reported earlier by Hanawa & Ota (1991) and Shabalovskaya & Anderegg (1995).Concentrations of Ni2+ ions in distant organs were not different in rats implanted withNiTi and stainless steel devices as measured from spleen, brain, liver, muscle and kidney60 weeks after implantation (Ryhänen et al. 1999a). Putters et al. (1992) concluded thatNiTi is biocompatible after studying the effects of NiTi, pure nickel and pure titanium infibroblast cultures. They found nickel to inhibit cell mitosis and NiTi to behave liketitanium. Contradictory results were obtained in a study of NiTi, titanium, Co-Cr-Mo, Ti-6Al-4V and stainless steel on fibroblasts. NiTi was found to have equally deleteriouseffects as Co-Cr-Mo on the direct contact of cells to material and was thereforeconsidered cytotoxic (Assad et al. 1994).

2.2.2.2 Biocompatibility studies of NiTi in vivo

Castleman et al. (1976) made experiments with dogs and found a thicker fibrous capsulesurrounding a NiTi implant than were found around other implants composed of Co-Cr-based alloys (Castleman et al. 1976). They concluded that this was caused by moreabundant metal dissolution from the NiTi implant.

On the other hand, a review of in vivo research reports on NiTi covering more than adecade disclosed no allergic reactions, no traces of alloy constituents in the surroundingtissue and no corrosion of implants (Shabalovskaya 1996). Takeshita et al. (1997) studiedthe response of rat tibiae to NiTi, comparing it to Ti-6Al-4V and stainless steel. Theyfound that the number and area of bone contacts was low around NiTi implants, but thethickness of contact was equal to that of other implants.

Poor results were reported in a study on the biocompatibility of NiTi in rabbit tibia,where NiTi, vitallium, pure titanium, austenite-ferric stainless steel and stainless steelscrews were implanted for 12 weeks. NiTi had no close contacts, and osteoblasts weredisorganised and showed scant osteonectin synthesis. The study, however, had severalweaknesses. Firstly, the authors used type III collagen antibody, which is definitively notuseful in measuring bone matrix formation. Secondly, the use of antibodies directedagainst mouse and goat epitopes in rabbit is slightly questionable. Lastly, the samplenumber (n=6) was too low for statistical analysis (Berger-Gorbet et al. 1996).

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In a recent study by Fili et al. (2001), NiTi implants were examined. The resultsdisclosed no irritation or adverse reactions in the human body against NiTi-basedimplants during a study period of 12 months. Their X-ray photoemission spectroscopy(XPS) studies indicated that passivation and reoxidation of NiTi prevents the formation ofoxidised nickel particles (Fili et al. 2001). Nickel oxides dissolve easily from the implantand may cause a harmful reaction in the surrounding tissue.

NiTi is non-toxic to muscle and perineural tissue, and the fibrous capsule was found tobe equally thick around all materials (Ryhänen et al. 1998). NiTi had no negative effecton bone formation, remodelling or consolidation of osteotomies when compared tostainless steel in a 60-week study with intermedullary implantation in rats (Ryhänen et al.1999a). Normal new bone formation was seen in rats at 26-week follow-up afterperiosteal implantation (Ryhänen et al. 1999b). NiTi was shown to have similar effects oncell-to-metal adhesion and bone formation at the end of a 26-week follow-up compared tostainless steel and Ti-6Al-4V.

Because of their good biocompatibility and corrosion resistance, titanium alloys,titanium alone, stainless steel, Co-Cr-based alloys and NiTi have been successfully usedas biomaterials (Kim & Johnson 1999). The tendency of NiTi to be covered by a titaniumoxide layer with only traces of nickel being exposed is considered to be responsible forthese good results. However, the good corrosion resistance of NiTi was challenged in areport of NiTi superelastic arch wire applied in the oral cavity (Yokoyama et al. 2001).Both NiTi and stainless steel showed corrosion after 60 weeks of intermedullaryimplantation in rats (Ryhänen et al. 1999a).

Taken together, there is still need for experiments to prove biocompatibility of NiTi.However, the growing number of studies implies that NiTi is well tolerated also inorthopaedic applications.

2.2.3 Surface of implant

The major problem associated with the currently used implants is due to inadequateimplant-tissue interface properties. The integration of load-bearing implants, such as hipand knee prosthesis and dental implants, into surrounding tissue is important. Both in vivoand in vitro studies have shown that implant surface topography may affect epithelial andconnective tissue behaviour. Based on these observations, a mathematical theory of idealsurface pit morphology, dimensions and densities of biomechanical significance wasformulated (Hansson & Norton 1999). Another theoretical analysis concluded that if thegeometric form of surface roughness is held constant, then the peak elastic stress dependson the form rather than the size of roughness (Skalak & Zhao 2000). However, thesemathematical models are frequently used in practice.

There are several in vitro studies that show how the physical, chemical andbiocompatible properties of implant surface can be improved. Human plasma fibronectincovalently immobilised to NiTi surface improved the attachment of cells (Endo 1995).

Coating of the implant surface is not always beneficial. The problem with titanium-based alloys is that the formation of TiO2, according to the equation Ti + 2H2O -> TiO2 +4H+ + 4e-, reduces pH at the titanium/coating interface. If the coating is composed of

30

hydroxyapatite, it dissolves and leads to detachment of the hydroxyapatite coating (Sousa& Barbosa 1996). The importance of surface treatment in NiTi was clarified in a studywhich showed that, depending on the treatment, the nickel surface concentration variedbetween 0.5% and 30%. A hydrogen peroxide-treated NiTi implant was found to beslightly more toxic than pure nickel. Autoclaving in water or steam influenced the NiTisurface in such a way that pretreated implants had no toxic effects on rat splenocytes(Shabalovskaya 1996).

Stanford et al. (1994) compared implant sterilisation methods using UV irradiation,autoclaving, ethylene oxide gas and plasma cleaning before culture with rat calvariaosteoblasts. They tested the effect of these procedures on three different surfaceroughness areas of pure titanium implants prepared using carbide paper 600-grit surfacepolishing, sand blasting with diamond paste, or polishing to mirror-like finish. Titaniumsurfaces treated with plasma glow discharge showed increased osteocalcin expression andALP activity with increasing surface smoothness. The other methods showed no similarrelationship between the method of sterilisation and surface roughness. Unfortunately,they did not measure the topography of these differently treated surfaces (Stanford et al.1994). It would be interesting to compare surface topographies to the above results.

Bordji et al. (1996a) tested the effects of three different surface treatments on 316Lstainless steel. All the treatments improved the wear and corrosion resistance of the alloy,but one of them, low-temperature plasma nitriding, impaired fibroblast and osteoblastproliferation and protein synthesis. In another study done using the titanium alloys Ti-6Al-4V and Ti-5Al-2.5-Fe, the same authors showed that fibroblasts and osteoblastsgrown on untreated or glow-discharged nitrogen-implanted alloys did not show signs ofimpaired growth. Again, nitriding treatments of the alloys clearly reduced cellproliferation and protein synthesis (Bordji et al. 1996b). When these two studies are takentogether, it seems that nitriding treatments of alloys may improve their physical propertiesbut not their cytocompatibility.

Implant surface roughness has an effect on cell orientation. Eisenbarth et al. (1996)found that fibroblasts orient along the long axis of the grooves on pure titanium and Ti-6Al-4V implants. The choice of cells used in the studies seems to affect the results.Bruinink & Wintermantel (2001) found that, in a rat bone marrow cell culture onpolystyrene with manually carved grooves, single cells oriented along the long axis of thegrooves. Further, when cells formed clusters, they still oriented along the grooves.However, when the same experiment was done with an osteoblastic cell line MC3T3-E1,cells did not orient themselves along the grooves.

In vivo studies of different surface characteristics on biocompatibility have been done.The effect of the three-dimensional structure of an implant surface was tested with adesign to compare porous NiTi and coralline hydroxyapatite in rabbit frontal boneimplantation. Porous NiTi has an open structure enabling the ingrowth of bone. Thisallows stable fixation of the implant to bone. The authors claimed that porous NiTi issuitable for craniofacial applications, since no harmful effects were noted and boneingrowth was good. This study, however, included only 6 rabbits (Simske & Sachdeva1995).

In a recent study on 30 rats, porous titanium mesh coated with Ca-P and filled withallogenic material (rat bone marrow cells) was used to induce ectopic bone formation.Interestingly, control implants without allogenic material did not induce bone formation.

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Only the implants containing allogenic cells induced ectopic bone formation (Vehof et al.2000).

The biocompatibility of RGD-peptide-coated titanium and gold-coated titaniumimplants was studied by implanting them in femoral canals of 23 rats. RGD-peptide has ahigh affinity to integrin α5β1 and is a common adhesive motif found in proteinsthroughout the body, including osteopontin, osteonectin and bone sialoprotein. RGD-peptide coating induced the formation of a thicker and more continuous coat of new bonethan gold-coated implant (Ferris et al. 1999).

Abron et al. (2001) compared a titanium implant of ideal pit density and surfacemorphology with a non-ideal surface with low pit density and with machined titanium ina rat tibia osteointegration model. The implantation time was 3 weeks and the resultsshowed a significant difference in the bone-implant contact in favour of the ideal surface.The authors defined the surface roughness measures with an AFM.

The surface characteristics seem to be important factors in biocompatibility. Asliterature sited here shows, there are many variations and possibilities to improve tissue-implant interface properties.

3 Aims of the study

New implant materials need to be tested properly before use in patients. Nickel titaniumshape memory alloy (NiTi) has unique damping properties, thermal shape memory andsuperelasticity properties not provided by any other implant alloys. These characteristicsmake it potentially useful for orthopaedic applications. A lot of interest has been directedtowards bone morphogenetic proteins (BMP). There have been several studies of surgicalimplants with BMP coating or administration. We hypothesised that bone induction couldbe utilised to improve the biocompatibility of implants. Based on a literature analysis,bone cells tolerate NiTi. We further hypothesised that relevant biological tests could bedeveloped to demonstrate its biocompatibility.

The aims of this work were:1. To study whether decalcified reindeer antler could be used as an implant material and,

especially, to find out if it contains BMPs beneficial for osteoinduction.2. To further develop the ectopic bone formation model for studying the effects of NiTi

on bone formation in vivo.3. To evaluate the effect of NiTi on viability, cytokine production and attachment of

osteoblasts.4. To evaluate the response of osteoblasts to NiTi implants with different surface

roughness values.

4 Materials and methods

4.1 Decalcified bone materials (I, II)

One-month-old, four-month-old and cast reindeer antlers and rat femurs were crushed inan ultracentrifugal mill (Retsch ZM100, F.Kurt Retsch GmbH & Co., Haan, Germany)with liquid nitrogen cooling to produce grains 0.5 ± 0.1 mm in diameter. Fat wasextracted with 1:1 chloroform:methanol mixture for one hour at room temperature withcontinuous stirring. The particles were decalcified in 0.6 N HCl for 24 hours at +4 °Cwith continuous stirring. Finally, the particles were washed with sterile water byrepeating the washing step several times. The particles were lyophilised and stored insterile vials at -20°C. For study I, the decalcified matrix was packed into gelatin capsules.In addition, for study II the test materials were placed in gelatin capsules (size no. 4,Orion, Seinäjoki, Finland) together with rat bone matrix powder.

4.2 Test materials (II-V)

Study II. The materials tested were vacuum-melted, drawn and fully annealed NiTi (54%nickel by weight, 46% titanium by weight, NiTi Development Co., Fremont, Ca, USA),AO/ASIF stainless steel (Synthes GmbH, Switzerland) and AO/ASIF Ti-6Al-4V alloy(90 % titanium by weight, 6 % aluminium by weight, 4 % vanadium by weight, SynthesGmbH, Switzerland). The surface of stainless steel was electrolytically polished, whereasthe surfaces of NiTi and Ti-6Al-4V samples were supplied in a mechanically groundcondition. Identical cylindrical implants 1.8 mm in diameter and 6 mm in length wereprepared from wire by cutting. The cylindrical implants were filled with decalcified ratbone matrix packaged into gelatin capsules.

Study III, V. The materials tested were vacuum-melted, drawn, and fully annealed NiTi(56 wt % Ni, 44 wt % Ti, Af=-10° C; Unitek, Monrovia, Ca, USA), stainless steel AISI316 LVM (12 % Ni, 18 % Cr, 68 % Fe, 2 % Mo; Sandvik, Sweden) ASTM Grade 2commercially pure titanium (TISTO GmbH; Düsseldorf, Germany) and commercially

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pure nickel (nickel plating grade used in electrolysis). Metal test discs, 5 mm in diameterand 3 mm thick, were produced from rods with a turning machine. The discs were highlypolished on one face.

Study IV, V. The materials tested were NiTi (56 % wt Ni, 44 % wt Ti, Af=-10° C;Unitek, Monrovia, Ca, USA) and Ti alloy (90.5 % wt Ti, 6 % wt Al, 2.2 wt % Mo, 1.3 wt% Cr, Institute of Light Metals, Moscow, Russia) vacuum-melted, drawn and hot-rolled.NiTi was hot-rolled at +950 °C. One set of Ti alloy specimens was hot-rolled at +850 °C(TiI) and the other at +1050 °C (TiII). The surface of the alloy discs had three differentroughness grades produced by mechanical grinding with stone N80 followed by polishingwith SiC sandpapers 240, 320, 400, 600, further with sandpapers 800, 1200 and finallywith a polishing cloth, the NiTi alloy having an additional treatment with a rubber wheelbefore polishing. The rubber wheel treatment was done to obtain surface as equal as thetitanium alloy had.

The test materials were all washed in an ultrasonic vibrobath, degreased with 70 %ethanol for 10 minutes and autoclaved at +120° C for 20 minutes before use.

4.3 Molecular biology methods

4.3.1 Isolation and characterisation of reindeer BMP-3b cDNA (I)

cDNAs were generated by RT-PCR with AMV reverse transcriptase (Finnzymes, Espoo,Finland) from poly(A) RNA of young antler tissue using oligo(T) primers. The cDNAswere amplified in PCR with degenerate oligonucleotide primers derived from the highlyconserved carboxy-terminal region of the BMP family to produce cDNAs related toknown BMPs. The 5’ and 3’ ends of cDNA were characterised with the 5’/3’ RACE kit(Boehringer Mannheim). The PCR products were cloned into pGEM®-T, and clones of aknown size insert were sequenced with the Perkin Elmer Applied Biosystems BigDyeterminator cycle sequencing kit and the ABI PRISM™ apparatus. Homology comparisonanalyses were performed with BlastX and BlastN programs, and prediction of proteinstructure was made using BlastP (Altschul et al. 1990).

4.3.2 In situ hybridisation (I)

In situ hybridisation can be used to assign the cellular location of gene expression.Digoxigenin-labeled single-strand RNA probes were prepared using the DIG RNAlabelling kit (Boehringer Mannheim). The BMP-3b probe was generated from a BMP-3b0.35 kb fragment in the pGEM®-T vector. Tissue sections of 5 µm from young reindeerantler were rehydrated with descending concentrations of ethanol, permeabilised with 20µg/ml of RNase-free proteinase K and prehybridised at +42 °C with prehybridisationbuffer (4 X SSC and 50% formamide). In situ hybridisation was carried out at +55 °C for16 hours in hybridisation buffer (40% formamide, 10 % dextran sulphate, 1% Denhart’s

35

reagent, 4 X SSC, 10 mM DTT, 1 mg/ml denatured salmon sperm DNA andapproximately 0.1 µg/ml of labelled antisense RNA probe). Posthybridisation washeswere done at 55 °C with stringent SSC washes. Hybridised probes were immunologicallydetected with an antibody to Digoxigenin conjugated to alkaline phosphatase enzyme(Boehringer Mannheim). Controls included hybridisation with the sense (mRNA) probe,RNAse A treatment (20 µg/ml) before hybridisation and use of neither antisense norantidigoxigenin antibody. None of the three controls showed positive signals.

4.3.3 Apoptosis detection (III)

Apoptosis detection methods can be used to study the apoptotic mode of cell death in cellcultures or in tissues.

4.3.3.1 DNA laddering

The cells on discs were washed with warm sterile PBS, and genomic DNA isolation andanalysis were performed with the TACStm DNA laddering kit (R&D Systems,Minneapolis, MN, USA). Cultures of six discs per test material produced approximately 1µg of genomic DNA, which was run on a 1.2 % agarose gel containing 0.06 mg/ml ofethidium bromide in 1 x TAE buffer at 100 V for 2 hours. Apoptotic DNA fragmentsconsist of multimers of 180-200 base pairs and appear as a DNA ladder. DNA of non-apoptotic populations of cells has high molecular weight and does not migrate far into thegel.

4.3.3.2 Terminal deoxynucleotidyl tranferase (TdT)-mediated dUTP nick end labeling (TUNEL)

TUNEL assay (Gavrieli et al. 1992) was used for the detection of apoptosis in PFA-fixedcells (TACStm TdT kit, R&D Systems, Minneapolis, MN, USA). TdT synthesisesfluorescein-labelled dUTP at the 3’-OH ends of the broken DNA strands, which areabundant in apoptotic nuclei. To confirm the nuclear morphology, the cells wereincubated with the DNA-binding fluorochrome Hoechst 33258 (1mg/ml stock diluted1:1000 in PBS, Sigma Chemical Co.) for 10 minutes at room temperature. From eachdisc, six randomly chosen areas (0.849 mm2) were viewed for apoptotic cells under afluorescence microscope (Nikon Eclipse E600, Nikon, Japan) with a 10 x objective, NA0.25 (Nikon, Japan).

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4.4 Cytotoxicity test (III, IV)

The cells on discs were stained with a LIVE/DEAD®Viability/Cytotoxicity kit(Molecular Probes, Eugene, Oregon, USA). The optimal concentration of calcein dye was1 µM and that of ethidium homodimer-1 (EthD-1) dye 0.1 µM. The samples wereincubated for 15 minutes at 37 °C and viewed under a fluorescence microscope. Deadcells (stained red) and live cells (stained green) were counted from six randomly chosenareas (0.849 mm2) on each disc. The cells were counted visually under a fluorescencemicroscope (Nikon Eclipse E600, Nikon, Japan) with a 10 x objective, NA 0.25 (Nikon,Japan), and the ratio of dead to live cells was calculated. Approximately 600 cells wereseen in each area, while the amount of dead cells per image was calculated as per 1000cells.

4.5 Enzyme-linked immunosorbent assay (ELISA) (V)

The ELISA method (Engvall & Perlmann 1971) is used for the determination of a proteinconcentration with an immunochemical reaction.

TGF-â1 and IL-6 cytokine concentrations were detected with the DuoSet human TGF-â1 and the Duoset rat IL-6 ELISA kits (R&DSystems, Minneapolis, MN, USA). LatentTGF-β1 in the sample media was activated with 1 N HCl. For absorbance measurements,the ELISA plate reader (Labsystems Multiskan® PLUS, Labsystems, Helsinki, Finland)was used.

4.6 Cell line (III-V)

The rat osteosarcoma cell line ROS-17/2.8 was a generous gift from G. A. Rodan (MerckResearch Laboratories, West Point, PA, USA). This cell line exhibits features close tothose of differentiated osteoblasts (Majeska et al. 1980, Thiede et al. 1988)

4.7 Animals (I,II)

The animal tests were performed with the approval of the ethical committee of theUniversity of Oulu. All aspects of animal care complied with the Animal Welfare Act andthe recommendations of the NIH-PHS Guide for the Care and Use of LaboratoryAnimals. All the animals used were male Sprague-Dawley rats from the LaboratoryAnimal Centre, University of Oulu (Oulu, Finland). The age of the animals was 3 monthsat the beginning of the experiments, and they weighed 350-450 g at that time. During theexperiment, the rats were housed in groups of 3-6 in Macrolon IV polycarbonate cages ina thermostatically controlled room at +20 °C and a under 12h/12h light/dark illumination

37

cycle. At the end of each experiment, the animals were sacrificed by CO2 suffocation andimplants were immediately removed and stored using the formalin fixation procedure.

4.8 Ectopic bone formation assay

All rats were anaesthetised with a fentanylcitrate (80 µg/kg) – fluanisone (2.5 mg/kg,Hypnorm®, Jansen-Pharmaseutica, Belgium) – Midazolam (1.25 mg/kg, Dormicum®,Roche, Basel, Switzerland) blend injection administered intraperitoneally. The hair wasshaven around the implantation site and the skin was sterilised by brushing it withchlorhexidin before the operation.

Study I: Decalcified matrix made of one-month-old, four-month-old and cast reindeerantlers and rat long bones (10 of each) was implanted, with two implants per rat. A singleskin incision of about 2 cm was made in midline between the scapulae. The implants wereplaced in gelatin capsules under the fascia of the right (allogenic implant or 4-month-oldantler) and left (cast antler or 1-month-old antler) latissimus dorsi muscle. After 3 weeksthe animals were euthanised and the formed ossicles were removed (all matrix samples,10 rats/sample). After 8 weeks, another set of animals were handled similarly (onlyallogenic and cast antler matrix, 10 rats/sample).

Study II: Ten NiTi, 8 stainless steel, and 8 Ti-6Al-4V implants with rat decalcifiedbone matrix packaged into gelatin capsules were implanted. All three implant materialswere placed under the fascia of the latissimus dorsi muscles of each rat (n=10). Tencontrol rats received gelatin capsules containing allogenic matrix without any metalimplant through a similar surgical operation. The animals were euthanised and the formedossicles were removed after 8 weeks.

The ossicle samples (I,II) were fixed in PBS-buffered neutral formalin for 7 to 14 days.After density measurements with pQCT, the implants were embedded in methacrylate(Technovit 7200), cut with a diamond saw, and micro-ground (Exakt Apparatebau GmbH,Germany). The ground samples with 5 µm sections (I) or 25 µm sections (II) were stainedwith the von Kossa and Masson-Goldner-Trichrome methods for histology andmorphometric light-microscopic computer-aided examination.

4.9 Methods of analysis

4.9.1 Peripheral quantitative computed tomography (pQCT) (I, II)

The total bone mineral density (BMD) of the ossicles was measured with pQCT (XCT920A, Norland Stratec Medizintechnik GmbH, Birkenfeld, Germany). Pixel size was0.145 µm2 and section thickness 1.25 mm. In study II, pQCT scans were taken 1mmapart from the implant based on a scout view image.

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4.9.2 Light microscopy (I, II)

The morphology and histology of the von Kossa and the Masson-Goldner-Trichromestained sections were examined under a Nikon Eclipse 600 light microscope (Nikon ,Tokyo, Japan) using either a 10 x/0.25, 20 x/0.50, or 40 x/0.75 plan fluor object (Nikon,Tokyo, Japan).

4.9.3 Bone histomorphometry (I, II) and digital image analysis (I-IV)

The real-color CCD camera-based digital image analysis system (MCID M4 v.3.0.rev.1.1., Imaging Research Inc., Ontario, Canada) consists of a color video camera (SonyXCT930P, Sony, Japan), a microscope (Nikon Optiphot II, Nikon, Japan) and a personalcomputer with a digitiser (Matrox Image 640 with CLD color board, ImagingTechnology, USA).

Study I: The proportional area of mineralisation in each implant was measured. VonKossa stained sections were analysed for black staining of mineralised tissue, and the areaof mineralisation was compared to the ossicle area.

Study II: Polarised light microscopy was used to distinguish between fibrotic tissueand bone. The proportional areas of fibrotic tissue and non-resorbed bone matrix powderwere compared to the implant area. The target area was cropped by excluding the newwoven bone, but including fibrotic tissue and non-resorbed initial rat bone matrix. Theproportional new bone area versus ossicle area and the proportional area of cartilageversus ossicle area were also examined.

Study III, IV: The number of focal contacts was measured with a digital imageanalyser (MCID M4 v.3.0.re.1.1, Imaging Research Inc., Ontario, Canada). The examinedregion of interest was 20117 µm2. Confocal microscope images were semiautomaticallysegmented on red color intensity, hue and saturation. The interactively defined focalcontacts were automatically counted from the region of interest.

4.9.4 Confocal laser scanning microscopy (III, IV)

The confocal laser scanning microscope consists of a 750 mW air-cooled argon-cryptonlaser (Omnichrome, Chino, CA, USA), a Leitz Aristoplan microscope, and softwareversion 1.05 (Leica Lasertechnik GmbH, Heidelberg, Germany). Rat osteosarcoma cellswere cultured in αMEM-FCS growth medium on different alloys for 48 hours, fixed with4 % PFA for 10 minutes at room temperature and permeabilised with 0.1 % Triton-X-100in PBS for 10 minutes on ice. For immunofluoresence staining, the samples wereincubated with primary monoclonal paxillin antibody (ZYMED Laboratories, Inc., SanFrancisco, CA, USA) for 45 minutes on ice. The samples were rinsed thoroughly severaltimes with PBS before the staining was completed with rhodamine-conjugated rabbitanti-mouse immunoglobulin secondary antibodies (DAKO, Glostrup, Denmark) for 30minutes on ice. To visualise the nuclei, the cells were incubated with the DNA-binding

39

fluorochrome Hoechst 33258 (1 mg/ml stock diluted 1:1000 in PBS, Sigma ChemicalCo.) for 10 minutes at room temperature. The focal contacts were studied under aconfocal microscope LSM 510 equipped with an inverted microscope Axiovert 100M anda 63 x (NA 1.2/w) water immersion objective (Zeiss, Göttingen, Germany). From eachsample disc, 6 frames were scanned with 1024 x 1024 frame size (pixel size 0.81 µm2).

4.9.5 Atomic force microscopy (AFM) (IV)

Atomic force microscopy was used to determine the surface roughness parameters of theNiTi and titanium alloys with different surface characteristics. AFM measurements wereperformed with an Explorer system (Thermomicroscopes, Sunnyvale, CA, USA) andSPMLabNT software ver. 5.01 Explorer AFM (Thermomicroscopes, Sunnyvale, CA,USA). The AFM Explorer system consists of a v-type cantilever, a CCD camera, laser,piezo positioners, an electronic control unit and a scanning and 3D graphics computer.The contact AFM method was used to survey the material surface. The probe tip,mounted on the cantilever, scanned across the sample surface in direct physical contact.As the scanning proceeded, varying topographic features caused deflection of thecantilever. This motion of the cantilever as it applies force to the sample, was used in afeedback loop to control the Z piezo, and the constant cantilever deflection wasmaintained. The scanning parameters were adjusted according to the surface topography.The size of the scanned area was100 x 100 µm and the resolution of image was 400 x 400pixel.

4.9.6 Statistical analysis (I-V)

Mean values and standard deviations were computed. Analysis of variance (ANOVA) andStudent’s t-test were utilised to assess the level of significance of the differences betweenthe experimental groups. All statistical analyses were performed with commercialsoftware (Origin 5.0, Microcal Software, Inc., Northampton, MA, USA). Averages(mean) and standard deviations (SD) are expressed in the tables and figures.

5 Results

5.1 Bone induction capacity of decalcified reindeer antler matrix (I)

5.1.1 Isolation and characterisation of BMP-3b cDNA (I)

Degenerate PCR was used to produce a pool of sequences of related BMPs. The firstprimers were designed from predicted homologues of the BMPs’ mature region cysteinesnumbered 4-5 and 6-7. This PCR produced sequences of approximately 100 base pairs.The second primer set was designed from the predicted human BMP-3b potentialglycolysation site in the precursor region of the BMP-3b and from the sequenced reindeermature peptide region cysteines 4-5. PCR with these primers produced 1050 base pairs ofthe BMP-3b sequence. 5’RACE produced some more nucleotides, but the starting codonand the 5’ untranslated region were not solved. We were able to produce 322 nucleotidesof the 3’ untranslated region. Overall, the produced cDNA sequence contained 1324nucleotides of the coding sequence for 441 amino acids.

5.1.2 Sequence analysis

A sequence comparison with human BMP-3b showed 91 % nucleotide homology in themature region and 86 % in the precursor region. The amino acid level homologies were90 % and 84 %, respectively. The partial coding sequence of reindeer BMP-3b wasdeposited in the GenBank under accession number AF300813.

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5.1.3 In situ hybridisation

Tissue distribution studies in the antler were made with in situ hybridisation. In situhybridisation with the BMP-3b probe showed that, in 1-month-old antler, BMP-3bmRNA is expressed in osteoblastic cells surrounding the capillaries in the centre of theantler. Less intense staining was seen in cells of the chondrogenic region farther from theantler centre.

5.1.4 Ectopic bone formation assay of antler matrix

Ectopic bone formation assay was used to study the bone induction potential of differentagents in live animals. We compared allogenic bone matrix with different stages of antlermaturation. None of the test animals showed any signs of inflammation when assessedvisually. Round ossicles were already seen in the 3-week implantation groups, but the 8-week cast antler ossicles showed irregular shapes and were softer than the same matrix inthe 3-week implantation. Histological evaluation of samples stained with von Kossa andtoluidene blue counterstain revealed differences in the ossicle morphologies of thedifferent matrix preparations. The ossicles of the allogenic matrix showed the phenotypeof endochondral ossification, but the antler matrix-induced ossicles expressedmesenchymal condensation similar to intramembranous ossification. Mineralisingnodules were seen around the capillaries in cast antler ossicles. The implanted antlermatrix did not induce ectopic ossification to the same extent as the rat bone matrix.During the 3-week induction period, there was a significant difference in BMD when theallogenic and antler matrices were compared. In addition, the ossicles induced by castantler matrix had significantly higher mineral density than the other two antler matrix-induced ossicles. When a longer implantation period was used, the allogenic matrix hadhigh BMD, while the 8-week ossicles of antler matrix did not have adequatemineralisation for x-ray attenuation to be measured with pQCT.

The histological von Kossa staining of the accumulated mineral was in line with theBMD. The proportional mineralised area in the implants of the allogenic matrix at 3weeks was 3.7±2.1 %. The proportional mineralised area was significantly smaller(p<0.005) in all antler matrix implants. When the four-month (0.25±0.17 %) and one-month (0.08±0.05 %) antler matrix implants were compared to the cast antler matrix at 3weeks (0.65±0.21 %), the differences were significant (p<0.05 and p<0.01, respectively).The proportional mineralised area of cast antler at 8 weeks was significantly smaller(1.2±0.01 %) compared to the allogenic matrix (11.6±0.1 %, p<0.01).

The measurement of the proportional amount of cartilage showed that there was nonormal cartilage in any of the antler matrix induced ossicles. Instead, the original matriximplant material with membranotic ossification, mesenchymal cell condensations andcalcified nodules were observed. In the 3-week allogenic implant, the proportion ofcartilage was 2.4±0.2 % of the total area.

The above observations of antler studies are shown summarised in table 2.

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Table 2. Comparison of different decalcified matrix patterns of bone induction.

5.2 Biocompatibility studies of NiTi (II-V)

5.2.1 Ectopic bone formation assay (II)

Ectopic bone formation assay was used to study the effects of implant materials on boneformation. After 8 weeks, advanced endochondral bone formation was evident. Most ofthe ossicles were filled with new woven bone, and there were very few decalcified boneparticles left. A very close contact between the implant and the new woven bone withoutfibrotic material was observed in some areas of two Stst samples, two Ti-6Al-4V samplesand one NiTi sample. BMD in the control group was 350±69 mg/cm3. The values ofNiTi, Stst and Ti-6Al-4V were 360±87 mg/cm3, 285±43 mg/cm3 and 293±73 mg/cm3,respectively.

Histomorphometric studies showed some differences between the tested materials inview of bone induction efficacy. The mean proportion of fibrotic tissue and bone matrixpowder was higher in the Ti-6Al-4V than in the other two alloys tested. The proportion ofcartilage was higher in the NiTi group (0.16±0.09) than in the Ti-6Al-4V group(0.05±0.04) (p<0.05). The proportion of cartilage in the control group was low. Theamount of new bone, measured as an area proportional to the area of the ossicle was highin the control group. Instead, the Stst group had significantly less new bone compared tothe controls (p<0.05).

The results of the ectopic bone formation assay are shown in table 3.

Table 3. Comparison of different implant materials for bone formation.

Qualifiers Allogenic Cast antler 4-month antler 1-month antler

BMD Dense Intermediate Sparse Very sparse

Mineralization area Large Small Very small Very small

Endochondral ossi-fication Evident Not detected Not detected Not detected

Qualifiers Allogenic NiTi Ti-6Al-4V Stainless steel

BMD Dense Dense Intermediate Intermediate

Fibrotic tissue area Not detected Intermediate Large area Intermediate

Cartilage area Little Large area Very little Intermediate

Bone area Large area Intermediate Intermediate Little

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5.2.2 Surface roughness (IV)

The surface roughness of materials in study IV was assessed with AFM. The averageroughness (Ra) values are expressed in table 4.

Table 4. Average surface roughness of implant materials (nm) mean ± 1 SD.

5.2.3 Cell viability (III, IV)

During the 48-hour culture period, cells reached confluency in all materials with asmooth surface. Cells cultured on NiTi survived, as did those on titanium. The cell deathrate was higher for cells cultured on stainless steel (p<0.05) and nickel (p<0.01)compared to NiTi and titanium.

When optically examined, the cells cultured on sandpaper-polished surfaces seemedlarger than those on smooth surfaces. All cultures, except TiII80 group, reached completeconfluency in 48 hours. The cytotoxicity test showed cell viability to be significantlybetter for the roughest NiTi and TiI surfaces than the other surfaces in the test groups. Thecell death rates on different materials are presented in table 5 and in figure 4.

Table 5. Dead cells/1000 cells cultured on surfaces with different chemical compositionsand surface roughness values. Mean ± 1 SD.

Materials 1200 600 80

NiTi 95±41 155±20 362±209

TiI [Ti-6Al-2.2Mo-1.3Cr(850° C)] 304±310 1428±173 1479±38

TiII [Ti-6Al-2.2Mo-1.3Cr(1050° C)] 339±39 436±10 732±49

1200, 600, 80 = SiC -sandpaper-polished, the number indicates the grade of paper used for finishing.

Materials Smooth 1200 600 80

NiTi 2.4±3.6 22±26 23.1±27.9 11.9±21

TiI [Ti-6Al-2.2Mo-1.3Cr(850° C)] 26.6±26.7 33.6±37 14.8±21.3

TiII [Ti-6Al-2.2Mo-1.3Cr(1050° C)] 25.1±43.3 22.8±28.3 33.5±29

Pure nickel 30.8±20.9

Pure titanium 2.9±3.3

Stainless steel 12.7±17.5

Smooth = highly polished, 1200, 600, 80 = SiC-sandpaper-polished, the number indicates the grade of paperused for finishing.

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Fig. 4. Cell viability on different materials.

5.2.4 Apoptosis of cells (III)

The DNA laddering assay showed negative results. To confirm the low apoptosis rate, westained the cells with TUNEL assay. The results of the TUNEL assay were combinedwith the number of dead cells detected in the viability assay, and the percentage ofapoptotic cells out of all dead cells was calculated (Table 6).

Table 6. Ratio of apoptotic cells in cultures on different materials. Mean ± 1 SD.

5.2.5 Cell attachment (III, IV)

To find out the possible effect of NiTi on cell attachment, we determined the number offocal adhesions based on paxillin staining of cells. The cells grown on nickel showedfewer visualised focal contacts, and most of the cells were rounded and stained diffuselywith rhodamine-paxillin antibody. The difference in the number of focal contacts was

Materials Apoptotic cells/1000 cells % apoptotic cells/dead cells

NiTi 1.93±2.1 46

Titanium 2.98±1.97 62

Stainless steel 1.1.±0.96 5.3***

Nickel 0.62±0.65* 1.2***

*: p<0.05 to titanium, ***:p<0.001

0

10

20

30

40

50

60

70

80

Dea

d ce

lls/1

000

cells

Stst

Ti

Ni

NiTi

NiTi1200

NiTi600

NiTi80

TiI1200

TiI600

TiI80

TiII1200

TiII600

TiII80

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significant between the Ni group and the Ti group. NiTi 80 strongly stimulated theformation of focal adhesions. TiI was a better matrix for osteoblast attachment than TiII(table 7, figure 5). The focal adhesions of the cells grown on the roughest surfaceappeared to locate along the surface grooves. This phenomenon was also observed,though less prominently, on the other surfaces.

Table 7. Number of focal adhesions per frame on surfaces of different roughness. Mean ±1 SD.

Fig. 5. Cell attachment on the materials described as the number of focal adhesions.

Materials Smooth 1200 600 80

NiTi 744.6±427 485±343 460±272 611±325

TiI [Ti-6Al-2.2Mo-1.3Cr(850° C)] 657.6±355 423.4±222 269±177

TiII [Ti-6Al-2.2Mo-1.3Cr(1050° C)] 350±194 246.8±156 223±151

Pure Ti 462.5±362

Pure Ni 261.5±226

Stainless steel 335.8±239

Smooth = highly polished, 1200, 600, 80 = SiC -sandpaper-polished, the number indicates the grade of paper used for finishing.

0.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0

Foca

l adh

esio

ns/fr

ame

StstNiTiNiTiNiTi1200NiTi600NiTi80TiI1200TiI600TiI80TiII1200TiII600TiII80

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5.2.6 Detection of TGF-β1 and IL-6 cytokines (V)

To find out how growth factor production might change on different materials, wemeasured TGF-β1 and IL-6 concentrations with the ELISA method. There was quite a lotof intragroup variation, but overall, the rough NiTi surface showed the highestconcentrations of TGF-β1. In all sample groups, the IL-6 concentrations were too low tobe detected with the system used. Table 8 and figure 6 show the results of the TGF-β1ELISA measurements.

Table 8. TGF-β1 concentrations (ng/ml) in various culture media. Mean ± 1 SD.

Fig. 6. TGF-β1 concentrations of cell cultures on different materials.

Materials Smooth 1200 600 80

NiTi 1.29±0.32 1.16±0.55 1.2±0.69 1.48±0.17

TiI [Ti-6Al-2.2Mo-1.3Cr(850° C)] 0.86±0.19 1.07±0.38 0.96±0.6

TiII [Ti-6Al-2.2Mo-1.3Cr(1050° C)] 1.37±0.18 1.27±0.25 1.5±0.17

Pure Ti 1.21±0.22

Pure Ni 1.06±0.42

Stainless steel 1.25±0.21

Smooth = highly polished, 1200, 600, 80 =SiC -sandpaper-polished, the number indicates the grade of paperused for finishing.

0

0.5

1

1.5

2

2.5

TGF-

beta

1 ng

/ml

Stst

Ti

Ni

NiTi

NiTi1200

NiTi600

NiTi80

TiI1200

TiI600

TiI80

TiII1200

TiII600

TiII80

6 Discussion

6.1 Bone induction capacity of decalcified reindeer antler matrix (I)

6.1.1 Isolation and characterisation of BMP-3b cDNA

To search for new bone growth factors from rapidly growing reindeer antler, we useddegenerate oligonucleotide primers in the amplification of a cDNA pool generated from1-month antler. Degenerate PCR has been used before in the detection of severalmammalian bone morphogenetic proteins (Chang et al. 1994). This method enables thediscovery of unknown, but related genes belonging to the same large protein family.

We were able to identify most of the cDNA sequence of the BMP-3b, 1323 nucleotidescoding for 441 amino acids. Previously, BMP-2 and -4 cDNAs have been cloned fromantler of red deer (Feng et al. 1995). These cDNAs prove that antlers express severaldifferent messenger RNAs of the BMP family, which probably have roles inantlerogenesis. Unfortunately, we did not succeed in the amplification and sequencing ofthe 5’ end of BMP-3b cDNA. Very often, the 5’ ends of the coding sequences are rich inguanine and cytosine nucleotides. These nucleotides form three hydrogen bonds witheach other, which produces stable secondary structures. The expected size of the codingsequence would have been around 1430 nucleotides, as predicted from the sizes of the rat(1431) and human (1434) sequences.

6.1.2 Sequence analysis and in situ hybridization

The close sequence similarity, especially in the mature peptide region, with human BMP-3b was no surprise, since the BMP family has been well conserved throughout evolution.

BMP-3b shares more sequence homology with BMP-3 than with the other BMPfamily members. In a recent study, BMP-3 was found to inhibit the effects on BMP-2responsiveness on osteoblastic cell lines. The explanation for this may be that BMP-3

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signals through the TGF-β/activin pathway and thereby antagonise BMP-2 signalling.Homozygotic knockout mice, which lack BMP-3, survive without any skeletal changesobserved in embryos or neonates. In adult mice, the lack of BMP-3 doubles the trabecularbone mass compared to wild-type mice (Bahamonde & Lyons 2001). On the contrary,BMP-3b knockout mice have no phenotypic changes, probably due to compensation byother BMPs (Zhao et al. 1999). BMP-3b plays a role in intramembranous ossification(Ripamonti et al. 2000). BMP-3b expression correlates with the differentiation of ratcalvarial osteoblasts (Hino et al. 1999).

To study BMP-3b in antlerogenesis, we studied its localization in antlers using in situhybridisation. This method revealed BMP-3b expression mostly in mature cells in theantler centre near the capillaries. Unfortunately, we were unable to produce slices ofcalcified antler tissue. The study of more mature antlers would have given newinformation about BMP-3b expression. In addition, it would have been most useful todetect changes in BMP-3b expression over time by using in situ hybridisation at differentstages of antler maturation.

The role of BMP-3b in reindeer antler development remains to be solved, but thisgrowth factor seems to be expressed in tissues when they reach the mineralisation stage.

6.1.3 Ectopic bone formation assay of antler matrix

Ectopic bone formation assay has been used for several decades (Urist 1965) to study theeffect of different compounds on the induction of bone. Our approach to use materialpackaged in gelatin capsules makes the handling of the test materials easier. The use ofthe back of the animal as the implantation site causes minimal harm to the animal. Theectopic bone formation assay showed that decalcified antler matrix contains agents thatactivate the immune system of the rat, even though this activation is not very prominent.We saw signs of inflammation in the ossicles, together with normally calcified nodules.Our results clearly indicate that native material should be purified more extensivelybefore its use.

The rationale of using immunogenic matrix, such as reindeer antler, for osteoinductionis that the antler is a rich source of bone growth factors, possibly even some currentlyunknown members of the BMP family. Even though a large number of BMPs havealready been discovered, the most effective ones might not yet been found. It is quiteclear that growth factors to be used in clinical applications will be produced withrecombinant DNA technology, not by extraction from native sources. Agents can beengineered so that they have reduced immunological effects. The production of fullyhumanized proteins will reduce the need for immunosuppressive medication, which isneeded when allografts are used.

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6.2 Biocompatibility studies of NiTi

6.2.1 Ectopic bone formation assay (II)

We used the ectopic bone formation assay to study the effect of NiTi on endochondralbone formation. Since the allogenic bone matrix induces endochondral ossification, themethod can be used to monitor the effects of materials embedded into the allogenicmatrix on bone formation. We refined the method of ectopic bone formation assay tostudy the biocompatibility of NiTi. Implantation of NiTi together with rat bone matrixenabled us to monitor the effect of the alloy on bone formation. Ectopic bone formationassay of reindeer antler had disclosed that the allogenic matrix forms fully mineralisedossicles in 8 weeks. This was therefore considered a sufficiently long implantation time,although ossicles with metal implants did not reach full mineralisation within this time

For comparison, we chose metal alloys frequently used in orthopedic surgery, namelystainless steel and Ti-6Al-4V. This comparison showed that these alloys behave in a verysimilar manner. Only the area of cartilage was higher in NiTi ossicles compared to othermetal alloys. This may indicate that the formation of cartilage is faster in ossicles formedin the presence of NiTi than the other alloys studied. Since the control ossicles werealmost fully mineralized, they were expected to have a low cartilage content and highmineral density. To our surprise, NiTi ossicles showed mineral density equal to thecontrol ossicles, even though the bone area was smaller than in the controls. NiTi wascloser to Ti-6Al-4V than to stainless steel as far as bone mineral density and bone areawere concerned, but fibrotic tissue was scant compared to the Ti-6Al-4V group. On theother hand, these results might imply that bone formation and mineralization are moreeffective in Ti-6Al-4V ossicles, which leaves only a minor role for cartilage between theresorption of the decalcified matrix and the ossification of the new matrix.

6.2.2 Cell viability (III, IV)

The ROS-17/2.8 rat osteosarcoma cell line was chosen as a the model cell line forosteoblasts because of their well-differentiated osteoblast phenotype and their ability toundergo apoptosis (Ihbe et al. 1998). In long-term implantation, mineralisation of bone isan important factor for the success of implantation. The use of ROS-17/2.8 cells enabledus to monitor the effects of the implant material on the differentiation of osteoblasticcells. A relatively long culture time, 48 hours, was chosen to ensure nickel release. After48 hours, the release of nickel decreases rapidly, probably due to the titanium oxide layerthat is formed on the surface of the NiTi implant (Ryhänen et al. 1997). Cultures wereperformed using low cell numbers (5000 cells/disc) to keep the cells as a monolayerduring the 48-hour culture period. High cell densities reduce the sensitivity of cells tometal ions (Wataha et al. 1993). The number of viable cells was counted usingfluorescence microscopy. Flow cytometry could also have been used, but it would havenecessitated trypsinisation of cells to detach them from the implant surface. Due to thelow cell number and the loss of cells during the handling of the samples, this would

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probably have given too low cell yields. We calculated the number of both dead and livecells to compare their ratio. The high number of dead cells on nickel samples was nosurprise, since nickel is known to be toxic. On the contrary, NiTi showed a low ratio ofdead to live cells, as did pure titanium. We believe that this shows the goodbiocompatibility of NiTi alloy, especially because stainless steel had a slightly worseratio.

The other methods frequently used to assess the viability of osteoblasts are based onDNA synthesis with thymidine incorporation, the MTT test, and ALP expression. TheMTT test is based on the reduction of diphenyltetrazolium bromide salt by mitochondrialenzymes. Frequently, ALP activity is measured using a test in which the enzymehydrolyses p-nitrophenylphosphate. Alternatively, ALP can be stained histochemically.The measurement of ALP activity is considered to be a more sensitive method for thedetection of nickel-induced toxicity than the MTT test (McKay et al. 1996).

In the study on the effect of surface roughness on cell viability, the roughest NiTi andTiI surfaces were better tolerated than the other surfaces. The better results obtained withNiTi than the titanium alloy are probably due to nickel being the only released particlefrom the surface, while titanium alloy may also release several compounds of aluminium,chromium and molybdenum in culture conditions. The TiI 80 surface did not disturb cellviability, possibly because of the heat treatment temperature. The rank order of cellviability on the different materials was TiII<TiI<NiTi.

6.2.3 Apoptosis of cells (III)

Programmed cell death, apoptosis, results in natural wastage of cells. There are manypossibilities to detect apoptosis. These methods are based either on the demonstration ofnuclear morphology or on the measurement of the activity of certain cytoplasmicenzymes, caspases, which are needed in cell lysis.

Detection of apoptosis is widely used in biocompatibility studies of biomaterials. Ourstudies showed very low rates of apoptosis induced by the alloys studied, even thoughNiTi and titanium had higher apoptotic indices than pure nickel and stainless steel. ROS-17/2.8 cell cultures have been shown to have a lag phase of growth until 72 hours (Ihbe etal. 1998). This may be the reason for the low apoptosis in our study: because cultureswould have still been at the lag phase of growth. In addition, it probably explains why nosigns of apoptosis were seen using DNA laddering. If the cells were still in their ofexponential growth phase, with a very low apoptosis rate, the detection limit (20 %apoptotic cells) of the DNA laddering technique was probably not reached. Cell viabilitywas similar on rough and smooth surfaces. Therefore, in the study on the effect of surfaceroughness on cell viability, TUNEL assay was not used to measure apoptosis. The singleproblem in the detection of apoptosis is that there are many phases in apoptosis. Oneassay does not necessarily cover every apoptotic event. Because of this, it is possible thatapoptotic cells, which are in different phases, are not all detected in one assay. We tried toavoid these problems by staining the TUNEL-stained cells with a nuclear stain andthereby to double-check the nuclear morphology.

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We found it informative to calculate the ratio of apoptotic to dead cells. This parameteris useful for the determination of the way cells die on implant materials. Our resultsshowed that, on pure nickel, most of the dead cells had died through necrosis.

6.2.4 Cell attachment (III, IV)

Attachment of the bone matrix producing cells on the orthopedic implant is considered tobe important for good osteointegration. The focal adhesions are artificial attachment sitesof the cells in the sense that they have been only observed in vitro (Abercrombie &Ambrose 1958). A focal adhesion consists of a protein complex in which integrin formsthe cell membrane spanning compartment and several other proteins inside the cellrespond to cell motility signals (Gumbiner 1993).

We studied the attachment of ROS-17/2.8 cells on different alloys with differentsurface roughness values. To analyse the effect of the implant on cell attachment, focaladhesions of the cells were stained with paxillin antibody. The number of focal adhesionsper picture frame could be used as a measure of cell adhesion because the cultures wereconfluent at the time of assay. Another measure of adhesion would have been the numberof focal adhesions per cell. However, this was not used because, in confluent cultures,almost all frames looked alike in this respect. There were no remarkable differences in theappearance of the cells; only the cells cultured on nickel had some morphologicalchanges.

In our study, the cells cultured on NiTi had almost twice as many focal contacts asthose cultured on pure titanium. This was an interesting result, since the morphology ofthe cells and the measures of cell viability showed marked similarities between titaniumand NiTi. Hunter et al. studied osteoblast and fibroblast attachment on differentorthopaedic biomaterials and found no significant differences in the attachment ofosteoblasts to different materials (Hunter et al. 1995). Their scanning electron microscope(SEM) observations were in line with morphometric data: cells with high numbers offocal adhesions were well spread and flattened, while cells with a low number of focaladhesions were rounded and less spread. In our study, cultures on pure nickel had thelowest numbers of focal adhesions, and these cells were round. We conclude that cellattachment to NiTi surfaces is good and definitely better than attachment to pure nickelsurfaces.

The roughness of the surface affected cell attachment in such a way that the cellsoriented along the grooves on all of the three implant materials. This observationdemonstrates that ROS-17/2.8 cells exhibit features common to rat bone marrow cells andfibroblasts, which were used in studies by Eisenbarth (1995) and Bruinink &Wintermantel (2001). In addition, rough NiTi and TiI surfaces supported cell attachment.Any comparison of NiTi and titanium alloys is difficult because they had different surfaceroughness values. The attachment sites of cells on NiTi were numerous in all surfaceroughness groups. Titanium alloys with two different heat treatments showed distinctdifferences in the cytocompatibility of rough surfaces. There were significantly fewerfocal adhesions in the cells cultured on material heated at 1050° C than on material heatedat 850° C. The heat treatment may stress material surface and, consequenctly, decrease

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the ion release from the surface in culture. These ions and the compounds formed of themin the cells could be cytotoxic. Unfortunately, we do not have data about the chemicalsurface characteristics of these differently treated titanium alloys. However, it seems thatosteoblasts sense implant surface characteristics and that this affects their attachment.There are no reports on the effect of austenitic versus martensitic surface oncytocompatibility.

As a conclusion, the materials studied in the present work can be placed in thefollowing rank order as to cell attachment: TiII<TiI<NiTi. This order is the same as wasobserved in the cell viability assays.

6.2.5 Detection of TGF-β1 and IL-6 cytokines (V)

ELISA measurements of TGF-β1 showed high intragroup variance. However, differentpatterns in TGF-β1 concentration where induced by different materials. TGF-β1concentrations increased in proportion to the roughness of NiTi and peaked in the middleroughness group of the TiI implants, but reached the minimum value in the middleroughness group of the TiII samples. TGF-β1 is a potent mitogen for osteoblastic cells(Robey et al. 1987). Because TGF-β1 was produced in all cultures, except those on nickeland TiI surfaces, we conclude that these cells signalled of proliferation. In contrast toviability and attachment, TGF-β1 concentrations lead to the following ranking:TiI<TiII≈NiTi. Overall, rough and smooth NiTi implants were cytocompatible. Onereason for the good results obtained with the use of smooth NiTi implants might be thatthe smooth implants were prepared of austenite alloy. All the ranking orders used areshown in table 9.

Osteoblasts are cells that are located close to orthopaedic implants. Remodelling startswhen the cells close to the implant signal to osteoclasts to start resorption. IL-6 couldperhaps be used as a marker of this signalling. IL-6 promotes osteoclastogenesis(Roodman 1992). In the present study, no IL-6 was detected in the samples collected fromthe osteoblast cultures on different alloys and with different surface roughness values.This observation may be due to the low cell count rather than a lack of IL-6 expression.However, it seems unlikely that the relatively short (48 hours) incubation period wouldexplain the lack of IL-6, since other studies have shown that the cytokine response israpid in cell cultures (Shida et al. 2000).

Table 9. Ranking orders of NiTi, TiI and TiII found in this study.

Property Ranking order

Cell viability TiII<TiI<NiTi

Cell attachment TiII<TiI<NiTi

TGF-β1 production TiI<TiII≈Niti

7 Conclusions

The degenerate oligonucleotide primer PCR produced a BMP sequence with highhomology to human and rat BMP-3b. This cDNA was named reindeer BMP-3b. Theeventual osteoinductive properties of reindeer antler matrix were tested using a modifiedectopic bone formation assay. It was shown that decalcified reindeer antler matrix wasimmunogenic and induced slight mineralization. Reindeer antler is perhaps a rich sourceof various BMPs, but the matrix must be more purified in vivo. However, if the reindeerantler matrix had had clear osteoinductive properties, the combined use of matrix andNiTi might have been possible.

We successfully modified the ectopic bone formation assay, so that it could be used fora biocompatibility study of NiTi. The modification used is animal-friendly and easy toperform. This method can be used for screening of biomaterials for their effects on boneformation prior to clinical use in animals or humans. Ossicles associated with NiTiimplants had similar characteristics as those associated with Ti-6Al-4V and stainless steelimplants. Because no inflammatory reactions were observed in the ossicles andhistomorphometric analysis showed endochondral bone formation, NiTi can beconsidered to be biocompatible.

Compared with pure titanium, pure nickel and stainless steel, NiTi was a well-toleratedosteoblast culture surface. NiTi showed cell viability similar to that of pure titanium. Cellviability on stainless steel came close to that of nickel. The high number of focal contactsshowed that cells attached well to NiTi surfaces. Cells had fewer focal contacts evenwhen cultured on pure titanium.

Surface roughness affected the viability, focal contacts and orientation of ROS-17/2.8cells. Surface roughness was sandpaper-modified of three different alloys: NiTi and Ti-6Al-2.2Mo-1.3Cr, the latter of which was also subjected to two heat treatments (+850°and +1050° C). Cells grown on NiTi and rough titanium alloy with heat treatment at+850° C showed good viability. Similarly, the rough surface NiTi and titanium alloy withheat treatment at +850° C induced good cell attachment. Less rough NiTi and titaniumalloy surfaces with heat treatment at +1050° C showed slightly higher cell death rates andfewer focal contacts. Cells grown on the rough surfaces oriented along the long axis ofthe grinding grooves.

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Cytokine detection in the supernatant of cells cultured on alloys turned out to besomewhat problematic as to IL-6, but not as to TGF-β1, due to the low cell counts. Weconclude that the ELISA method used in this study can be used to screen cytokinesignalling of osteoblasts cultured on biomaterials.

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