BIOCOMPATIBILITY OF ORTHOPAEDIC IMPLANTS ON BONE FORMING CELLS

ANITA K A PA N E N
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 of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium A 101 of the Department of Anatomy and Cell Biology, on February 22nd, 2002, at 12 noon.

O U L U N Y L I O P I S TO, O U L U 2 0 0 2

Copyright 2002 University of Oulu, 2002

Reviewed by Professor Yrj Konttinen Doctor Jukka Lausmaa

ISBN 951-42-6606-4

(URL: http://herkules.oulu.fi/isbn9514266064/)

ALSO AVAILABLE IN PRINTED FORMAT ISBN 951-42-6605-6 ISSN 0355-3221 (URL: http://herkules.oulu.fi/issn03553221/) OULU UNIVERSITY PRESS OULU 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 2002 Oulu, Finland

Abstract
Reindeer antler was studied for its possible use as a bone implant material. A molecular biological study showed that antler contains a growth factor promoting bone formation. Ectopic bone formation assay showed that antler is not an equally effective inducer as allogenic material. Ectopic bone formation assay was optimised for biocompatibility studies of orthopaedic NiTi implants. Ti-6Al-4V and stainless steel were used as reference materials. The assay showed differences in bone mineral densities, with superior qualities in NiTi. The rate of endochondral ossification varied between the implants, NiTi ossicles had larger cartilage and bone areas than ossicles of the two other materials. The cytocompatibility of NiTi was studied with three different methods. Cell viability, cell adhesion and TGF-1 concentration were assessed in ROS-17/2.8 cell cultures. Cells grown on NiTi had better viability than cells grown on pure nickel or stainless steel. Cell attachment on the materials was studied with paxillin staining of focal contacts. The number of focal contacts was clearly higher in cells grown on NiTi than in cells grown on pure titanium, pure nickel or stainless steel. TGF-1 concentration was measured with ELISA. The results showed that there was only some minor variation between NiTi, pure titanium and stainless steel. Nickel showed a lower TGF-1 concentration. 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 on cytocompatibility. Three different surface roughness grades were compared in cell cultures on NiTi and titanium alloy discs. Titanium alloy was subjected to two different heat treatments, to compare the effects of the treatments on cytocompatibility. The studies showed that NiTi had a lesser impact on cell viability and attachment than titanium alloy. Further, rough NiTi was found to be a better tolerated surface than the others. In this study, heat treatment of titanium alloy at +850 C did not interfere with cell viability or attachment, as did the +1050 C treatment of the alloy. On the contrary, TGF-1 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 adhesion, cell death

To the cliffs of the Lake Saimaa

Acknowledgements
This work was carried out at the Department of Anatomy and Cell Biology and Biocenter Oulu, 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 and excellent sense of humour have been encouragement for me. He should be acknowledged as the best supervisor one can have. I am grateful to Professor Yrj 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 Ryhnen, MD, PhD, for introducing me to the interesting world of NiTi. You have showed me the way. I thank Anatoli Danilov, PhD, for teaching me the basics of shape memory metals. I appreciate him as a true intellectual, with a very warm heart. I thank my other co-authors, Joanna Ilvesaro, PhD, and Docent Petri Lehenkari, MD, PhD, for fruitful co-operation. Anne Kinnunen is acknowledged for her never-failing friendship. We have supported each other in this mad world of academics and shall continue to do so. In addition, to our surprise, we have been co-operating even outside the coffee breaks! I thank my first supervisor, Professor Kalervo Vnnen, MD, PhD, for introducing me into the scientific world. His research group of Bone Heads was the best possible place to learn research work. I thank Elli Birr, PhD, for the three years we shared with antlers. We certainly had a hard material and hard times, but we also shared the fascinating joy of discovery. I thank Maritta Perl-Heape, PhD, for mentoring me through these years. I thank the whole staff of the Department of Anatomy and Cell Biology. The years I have spent at the Anatomy have given me the inspiration to keep going, to move on. I also wish to thank Minna Vanhala, Marja Paloniemi, Merja Nissil and Pirkko Peronius for their skilful technical assistance. For the unforgettable moments of womanhood, I want to thank the girls of our office: Tuula Kaisto, Hinni Papponen and Marja Nissinen, PhD. Could there be a better contraceptive 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 of hard times at home to make me manage in the outside world. I guess, no matter how masochistic it might sound, this should be gratefully acknowledged. Finally, I want to express 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 person like 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 Farmos and the Pohjois-Pohjanmaa Fund of the Finnish Cultural Foundation. Oulu, January 2002 Anita Kapanen

Abbreviations
AFM AISI ALP AMV AO/ASIF As ASTM AuCd BlastN BlastP BlastX BMD BMP BMPR BSP CBFA-1 CDMP cDNA Co-Cr-Mo Cu-Al-Ni DNA DTT dUTP ELISA GDF HCl ICAM IL InTl Md Atomic force microscopy American Iron and Steel Institute Alkaline phosphatase Avian Myeloblastosis virus Arbeitsgemeinschaft fr Osteosynthesefrage/Association for the Study of Internal Fixation Austenite start temperature American Society for Testing and Materials Gold-cadmium alloy Basic local alignment tools, nucleotides Basic local alignment tools, proteins Basic local alignment tools, both nucleotides and proteins Bone mineral density Bone morphogenetic protein Bone morphogenetic protein receptor Bone sialoprotein Core-binding factor-alpha 1 Cartilage-derived morphogenetic protein Complementary deoxyribonucleic acid Cobalt-chromium-molybdenum alloy Copper-aluminium-nickel alloy Deoxyribonucleic acid dithiotreitol deoxyurasiltriphosphate Enzyme-linked immunosorbent assay Growth and differentiation factor Hydrochloride acid Intercellular adhesion molecule Interleukine Indium-tellurium alloy Highest temperature to strain-induced martensite

Mf Martensite finish temperature Mg Magnesium Ms Martensite start temperature MT Martensite transformation MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Ni Nickel NiO Nickel oxide Ni3S2 Nickel sulphide NiTi Nickel-titanium alloy OP Osteopontin OP-1 Osteogenic protein-1 PBS Phosphate-buffered saline PDGF Platelet-derived growth factor PFA Paraformaldehyde pQCT Peripheral quantitative computed tomography 5/3 RACE Rapid amplification of cDNA sequence ends RGD Arginine-glycine-aspartic acid RNA Ribonucleic acid RT-PCR Reverse transcriptase polymerase chain reaction ROS-17/2.8 Rat osteosarcoma cell line SMA Shape memory alloy SME Shape memory effect Stst Stainless steel alloy TdT Terminal deoxynucleotidyl transferase TGF-1 Transforming growth factor-1 Ti Titanium Ti-6Al-4V Titanium-aluminum(6%)-vanadium(4%) alloy TiO2 Titanium oxide Ti-6Al-2.2Mo-1.3Cr Titanium-aluminum(6%)-molebdenum(2.2%)-chromium(1.3%) TNF- Tumor necrosis factor- TTR Transition temperature range TUNEL Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling UV Ultraviolet Vgr Vegetal related

Definitions
Allograft Austenite Autograft Biocompatibility Material taken for grafting from another individual of the same species. The high-temperature (parent) phase of some metals, e.g NiTi. Material taken from the same individual. A general term used to describe the suitability of a material for exposure to the body or bodily fluids. The specific meaning is dependent upon the particular application or circumstances. A material intended to come into contact with biological systems. The ability of a material to perform an appropriate cellular response. A material to be transplanted into the body In case of SMA, the difference of temperature at which the material is 50% transformed to austenite upon unloading and 50% transformed to martensite upon stress. A medical device made of one or more biomaterials that is placed within the body. The low-temperature phase of some metals, e.g. NiTi. A lattice transformation involving shearing deformation and resulting from cooperative atomic movement Ability to guide bone formation on a material surface in a bony environment. Ability to induce bone formation in non-osseous tissue. Material with an ability to return to some previously defined shape or size when subjected to an appropriate stress procedure. An alloy whose fixed shape has been stored, which, after deformation by stress followed by stress release, reverts to its original shape.

Biomaterial Cytocompatibility Graft Hysteresis

Implant Martensite Martensite transformation Osteoconduction Osteoinduction Shape memory alloy

Shape memory effect

Superelasticity (pseudoelasticity) The ability of a material to fully recover its initial shape upon 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 their Roman numerals. I II III Kapanen A, Ryhnen J, Birr E, Vnnen K & Tuukkanen J (2002) Bone morphogenetic protein 3b expressing reindeer antler. J Biomed Mat Res 59:78-83. Kapanen A, Ryhnen J, Danilov A & Tuukkanen J (2001) Effect of nickeltitanium shape memory metal alloy on bone formation. Biomaterials 22:2475-2480. Kapanen A, Ilvesaro J, Danilov A, Ryhnen J, Lehenkari P & Tuukkanen J (2002) Behaviour of Nitinol in osteoblast-like ROS-17 cell cultures. Biomaterials 23:645650. Kapanen A, Danilov A, Lehenkari P, Ryhnen J & Tuukkanen J. Effect of metal alloy surface roughness on the viability of ROS-17/2.8 osteoblastic cells. Submitted Kapanen A, Kinnunen A, Ryhnen J & Tuukkanen J. TGF-1 secretion of ROS-17/ 2.8 cultures on NiTi bone implant. Biomaterials, in press.

IV V

Contents
Abstract Acknowledgements Abbreviations Definitions List of original publications 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Review of the literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1 Structure and function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2 Bone-forming cells: osteoblasts, osteocytes, lining cells . . . . . . . . . . . . . . 2.1.3 Remodelling cycle of bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4 Bone morphogenetic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Biomaterials in orthopaedics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 Shape memory metal NiTi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2 Cytotoxicity of nickel and titanium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2.1 Biocompatibility studies of NiTi in vitro . . . . . . . . . . . . . . . . . . . . 2.2.2.2 Biocompatibility studies of NiTi in vivo . . . . . . . . . . . . . . . . . . . . 2.2.3 Surface of implant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Aims of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Decalcified bone materials (I, II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Test materials (II-V) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Molecular biology methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1 Isolation and characterisation of reindeer BMP-3b cDNA (I) . . . . . . . . . . 4.3.2 In situ hybridisation (I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3 Apoptosis detection (III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3.1 DNA laddering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3.2 Terminal deoxynucleotidyl tranferase (TdT)-mediated dUTP nick end labeling (TUNEL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Cytotoxicity test (III, IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Enzyme-linked immunosorbent assay (ELISA) (V) . . . . . . . . . . . . . . . . . . . . . . 4.6 Cell line (III-V) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 Animals (I,II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

17 18 18 18 19 21 22 24 24 26 27 28 29 32 33 33 33 34 34 34 35 35 35 36 36 36 36

7 8

4.8 Ectopic bone formation assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9 Methods of analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9.1 Peripheral quantitative computed tomography (pQCT) (I, II) . . . . . . . . . . 4.9.2 Light microscopy (I, II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9.3 Bone histomorphometry (I, II) and digital image analysis (I-IV) . . . . . . . 4.9.4 Confocal laser scanning microscopy (III, IV) . . . . . . . . . . . . . . . . . . . . . . 4.9.5 Atomic force microscopy (AFM) (IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9.6 Statistical analysis (I-V) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Bone induction capacity of decalcified reindeer antler matrix (I) . . . . . . . . . . . . 5.1.1 Isolation and characterisation of BMP-3b cDNA (I) . . . . . . . . . . . . . . . . . 5.1.2 Sequence analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.3 In situ hybridisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.4 Ectopic bone formation assay of antler matrix . . . . . . . . . . . . . . . . . . . . . . 5.2 Biocompatibility studies of NiTi (II-V) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1 Ectopic bone formation assay (II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2 Surface roughness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3 Cell viability (III, IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.4 Apoptosis of cells (III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.5 Cell attachment (III, IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.6 Detection of TGF-b1 and IL-6 cytokines (V) . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Bone induction capacity of decalcified reindeer antler matrix (I) . . . . . . . . . . . . 6.1.1 Isolation and characterisation of BMP-3b cDNA . . . . . . . . . . . . . . . . . . . 6.1.2 Sequence analysis and in situ hybridization . . . . . . . . . . . . . . . . . . . . . . . . 6.1.3 Ectopic bone formation assay of antler matrix . . . . . . . . . . . . . . . . . . . . . . 6.2 Biocompatibility studies of NiTi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.1 Ectopic bone formation assay (II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.2 Cell viability (III, IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.3 Apoptosis of cells (III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.4 Cell attachment (III, IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.5 Detection of TGF-b1 and IL-6 cytokines (V) . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

37 37 37 38 38 38 39 39 40 40 40 40 41 41 42 42 43 43 44 44 46 47 47 47 47 48 49 49 49 50 51 52 53 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 orthopaedics vary a lot as to their source and nature, but they are expected to share some common properties. Biomaterials, including orthopaedic implants, should not cause host responses, such as tissue necrosis. Osteolysis, bone resorption and the formation of a thick fibrotic capsule indicate poor biocompatibility. The implant should have resistance against mechanical load, which is one of the basic properties of healthy bone. It would sometimes also be beneficial if the implant could stimulate bone formation. Some metals are suitable for load-bearing implants because of their mechanical strength and biocompatibility. An increase of implant wear, however, sometimes increases the surface area and the concentration of metal ions. It is therefore important that the mechanical properties of the implants and their surface are able to minimize ion leakage. Implants that stimulate bone formation include hydroxyapatite-based materials or auto-, allo- and xenografts of bone. Allo- and xenografts may lead to problem due to immunologic host reactions. Depending on the case, a combination of load-bearing metal and bone formation-stimulating material in the same implant might be better than a metal or bone-inductive implant alone. The experimental part of this project focused on the methods that can be used to study the 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 to induce bone formation was studied. The bone induction properties of antler were studied using molecular biological methods and ectopic bone formation assay. We optimized the ectopic bone formation assay to study the biocompatibility of NiTi. Further, cell culture studies were done to clarify the effects of NiTi on cell viability, attachment and cytokine release. The surface roughness effects on cells were also examined. Pure elements of the alloy, i.e. titanium and nickel, and some commonly used orthopaedic implant materials were 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 mechanical strength, shelter for internal organs and a place for blood formation and plays an important role in the calcium homeostasis of the body. Bone consists of intercellular matrix and bone-forming cells, i.e. osteoblasts, osteocytes, lining cells and boneresorbing cells, i.e. osteoclasts. The intercellular matrix is composed mostly of collagen type I as the major organic compound and inorganic components, such as calcium phosphate. Inorganic salts account for two thirds of the weight of bone and are responsible for the hardness of osseous tissue. There are two different types of bone: cancellous and cortical. Cancellous bone consists of irregular bars, or trabeculae, that branch and form a mesh, which is filled with bone marrow. Cancellous bone has very active metabolic functions due to its proximity to and extensive interphase with bone marrow. Cortical bone is compact and has protective and mechanical functions. In long bones, cortical bone covers the cancellous bone like a tube. Bones may develop via two mechanisms: intramembranous and endochondral ossification. Both types are active in embryonic development and in adult bone renovation. Intramembranous ossification occurs in flat bones of the skull. In the developing skull, mesenchymal tissue is strongly vascularised and some mesenchymal cells differentiate directly into osteoblasts. Mineralisation is initiated in the matrix vesicles that bud away from the plasma membrane of the osteoblast, similarly to cartilage mineralization (Hohling et al. 1978). Matrix vesicles are extracellular organelles, which contain proteinases, peptidases, and alkaline phosphatase (ALP) (Hirschman et al. 1983, Dean et al. 1994, Wuthier et al. 1985). Further, hydroxyapatite crystals are first seen on vesicle membrane, potentially serving as nuclei for subsequent bulk calcification of the extracellular matrix (Anderson 1969). Endochondral ossification typically occurs in long

19 bones. Mesenchymal cells first differentiate into chondroblasts to form a cartilaginous model, which is subsequently mineralised. Matrix-resorbing osteoclasts, which have differentiated from their heamotopoetic precursors, remove the mineralised cartilage, after which the mesenchymal osteoprogenitor cells differentiate into osteoblasts. They produce new bone, while the remnants of calcified cartilage form a supporting framework. Mineralization begins by the synthesis of components of collagen or noncollagenous proteins (Marks & Hermey 1996). Transcription factor CBFA-1 has a key role in bone formation (Ducy et al. 1997). CBFA-1 knock-out mice (mice without the Cbfa-1 gene) had no bone formation, because both endochondral and intramembranous ossification were eliminated (Komori et al. 1997, Otto et al. 1997). Antlers, the result of a peculiar form of endochondral bone formation, are bony cranial organs unique to the Cervidae family (Chapman D.I. 1975). Antlers differ from Bovidae horns in that they are cast every year and that antler growth takes place at the tip (Goss 1983b). Regenerating antler is covered by skin with thick, fine hair. Therefore, growing antler is called velvet antler. The antler grows by a process of modified endochondral ossification; that is, bone formation takes place in a preformed frame of cartilage, which is highly vascularised (Banks 1974). There is a zone of fibroblasts at the very tip of the antler, followed by layers of cartilage, calcified cartilage and ultimately bone, forming a gradient of progressive differentiation from the zone of active cell division at the tip to mineralised bone at the antler root (Suttie & Fennessy 1990). Superficial arterioles supply the 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 an interesting 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 been used for the production of BMP implantation material. The BMP fraction produced has been shown to be highly active (Viljanen et al. 1996). Three specific BMPs have been sequenced 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 found in 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 function is to produce new bone matrix, osteoid, and to mineralise it. During differentiation, the osteoblast goes through morphological and functional changes. The preosteoblast is flat and expresses transforming growth factor- (TGF-), which induces osteoblast cell proliferation (Noda & Camilliere 1989) (figure 1). In addition, osteopontin (OP) is expressed, even though its expression peaks later. The proliferating osteoblast expresses proteins needed in cell division, such as histones, c-fos and c-myc, but early also the main protein 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 cell becomes fully differentiated and cuboidal in shape. A differentiated osteoblast expresses osteopontin and a closely structurally related protein known as bone sialoprotein (BSP). Finally, during the mineralisation phase, the cell produces osteocalcin, a major noncollagenous protein in bone (Stein et al. 1996). Approximately 50-70 % (Jilka et al. 1998) of osteoblasts die trough programmed cell death, apoptosis, while the rest are embedded in the matrix or form a layer of resting cells on the bone surface. The embedded cells are called osteocytes, and they are responsible for the maintenance of bone. Osteocytes constitute a functional syncytium, which communicates through dendritic processes and gap junctions (Jones et al. 1993). This syncytium also includes bone-lining cells or osteoblasts, depending on whether the bone in question is resting or remodelling (Palumbo et al. 1990). Osteocytes may communicate with bone-resorbing osteoclasts. Both osteoclasts and osteocytes, when located in close contact with each other, contain antigen CD44, which is macrophage lineage specific, and osteopontin, which is an osteoblastic protein (Yamazaki et al. 1997). The osteoblasts that constitute a resting layer instead of dying through apoptosis are called lining cells. A lining cell is an inactive, postproliferative cell covering a bone surface that is undergoing neither bone formation nor resorption. Studies have shown that these cells can be reactivated into bone-producing osteoblasts (Chow et al. 1998). They are thought to be responsible for the activation of bone remodelling by producing cytokines and other signals, which activate osteoclasts. The osteocytic syncytium produces and transmits signals proportional to mechanical loading (Martin 2000a). Marotti et al. 1992 suggested that osteocytes can send an inhibitory signal to osteoblasts that reduces their rate of bone formation (Marotti et al. 1992). This hypothesis has been developed further by Martin 2000, who has described an osteocytic inhibitory signal produced in response to mechanical loading able to inhibit bone lining cells from activating 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, and binding of these factors to their receptors activates signal transduction pathways that finally lead to nuclear responses. These hormonal factors include estrogen and parathyroid hormone. Local factors, which affect directly the same cell or neighbouring cells, include bone morphogenetic proteins (BMPs) and their antagonists, noggin and chordin.
Preosteoblast TGF-, osteopontin Proliferating osteoblast Histones, collagen type I Maturating osteoblast Alkaline phosphatase Differentiating osteoblast Osteopontin, bone sialo prot. Mineralization Osteocalcin

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

21

2.1.3 Remodelling cycle of bone

Bone tissue is under constant reconstruction. Approximately 30% of bone mass is remodelled in a year. This is necessary for normal skeletal maintenance. Bone matrix is produced and mineralised by osteoblasts, as described in the previous chapter. Bone resorption is done by bone-resorbing cells, osteoclasts. Osteoclasts originate from the hematopoetic-macrophage lineage. Their mononuclear precursors use vascular routes to enter skeletal sites, where they fuse to become active, multinucleated osteoclasts. By a mechanism still unknown, osteoclasts are guided to appropriate sites to be resorbed. There are theories postulating that osteocytes, osteoblasts and bone-lining cells regulate the ionic flow between the syncytium and the extracellular fluid of bone. This communication network seems to influence two bone cell activities: strain-related adaptive remodelling and mineral exchange (Rubinacci et al. 1998). During bone resorption, osteoclasts go through several steps, including attachment to the bone surface, polarisation of the cell surface into three distinctive membrane compartments, formation of a sealing zone, resorption, final detachment and eventual cell death (Vnnen 1996). Resorption of bone leads to a release of the growth factors buried in the bone matrix, such as TGF-, BMPs and other factors that activate and recruit osteoblasts to form new bone at the resorption site. Osteoblasts, in turn, produce growth factors, such as BMPs and PDGF, which are embedded in the newly synthesised bone matrix, and cytokines, which modulate osteoclast activity, including IL-11, IL-6, IL-8 and TNF- (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 are identical to those taking place in the growth plate during development, with the exception that these events occur on a temporal rather than a spatial scale. Fracture healing may be viewed as consisting of four distinct responses occurring in different tissue compartments. They take place in bone marrow, cortex, periosteum and external soft tissue. In bone marrow, there is a rapid transformation of endothelial cells into osteoblasts (Brighton & Hunt 1991). The cortex may either directly re-establish itself (primary cortical healing) or form a callus, which is associated with responses in the periosteum and external tissue (secondary fracture healing). The majority of fractures heal by the mechanism of secondary fracture healing. In the periosteum, osteoprogenitor cells and undifferentiated mesenchymal cells contribute to the healing process by intramembranous and endochondral ossification. Intramembranous ossification occurs farther from the site of fracture, resulting in the formation of hard callus. Intramembranous bone is formed by osteoprogenitor cells of periosteum. Adjacent to the fracture site, bone is formed by endochondral ossification. Secondary fracture healing starts with the formation of a hematoma and inflammation. Inflammatory cells secreting cytokines, such as IL-1 and IL-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 bone formation. Finally, bone remodelling begins to take place.

22

2.1.4 Bone morphogenetic proteins

Bone morphogenetic proteins (BMPs) are a large family of secreted polypeptides with common structural features and diverse effects on tissue development (table 1). The alignment of amino acid sequences of BMPs indicates significant amino acid sequence identity in the carboxy-terminal region of the proteins. BMPs are synthesised within cells as precursor forms. They have a hydrophobic leader sequence, a long propeptide portion and a carboxy-terminal mature peptide domain. In the mature region, BMPs have seven conserved cysteines (figure 2). These cysteine residues are important for the threedimensional structure of the protein. Cysteine residues 1-3 and 4,6-7 form disulfide bonds in the BMP monomer, but the fifth cysteine residue bonds with the complementary residue in the other BMP monomer to produce a dimer (Griffith et al. 1996). After proteolytic cleavage of the leader sequence and the propeptide, the mature regions form dimers intracellularly (Hazama et al. 1995). Their role in bone remodelling and fracture healing is discussed briefly.
Leader 15-25 aa Y Propeptide Y Mature region Y CC CC C

RXXR

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

Among the various factors that could be used to facilitate fracture healing, the expression of bone morphogenetic proteins is thought to be important. During fracture repair, BMPs are needed for the initiation of cell differentiation. The further the differentiation 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, namely hypertrophic chondrocyte (Grimsrud et al. 1999). There are several preclinical animal studies 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 tibial nonunions of human patients with good results (Friedlaender et al. 2001).

23 Table 1. Bone morphogenetic protein superfamily.

BMP BMP-2 Major expression tissue Cartilage, bone Other names BMP-2a References Mammalian cDNA sequenced

Bovine, human, Wozney et al. 1988, mouse, rat, reindeer Sampath et al. 1990 Feng et al. 1994, 1997 Wozney et al. 1988, Takao et al. 1996 Bovine, human, rat

BMP-3 BMP-3b

Cartilage, bone, ovary Osteogenin Muscle, bone, brain, lung GDF-10

Human, rat, mouse, Cunningham et al. reindeer 1995 Hino et al. 1996, Takao et al. 1996 Kapanen et al. 2000 Wozney et al. 1988, Feng et al. 1995 Celeste et al.1990 Human, mouse, reindeer Bovine, human Bovine, human, mouse, rat Bovine, human, mouse, rat Human, mouse Human Human, mouse Human, mouse Human, mouse Human, mouse, bovine Bovine, mouse, human

BMP-4 BMP-5 BMP-6 BMP-7 BMP-8a,b BMP-9 BMP-10 BMP-11 BMP-12 BMP-13

Cartilage, bone, prostate Bone Cartilage Bone, kidney Testis, placenta Liver Bone, heart Neurogenesis Cartilage Cartilage

BMP-2b

Vgr-1 OP-1 OP-2, OP-3 GDF-2

Celeste et al.1990 Gitelman et al.1994 Celeste et al.1990 Simon et al. 1999 zkaynak et al. 1990, Zhao et al. 1996 Song et al. 1995 Celeste et al. 1995, Neuhaus et al. 1995 Celeste et al. 1995

GDF-7, CDMB-3 GDF-6, CDMB-2

Strom et al. 1994, Celeste et al. 1995 Strom et al. 1994, Dube & Celeste 1995, Chang et al. 1994 Strom et al. 1994, Chang et al. 1994

BMP-14 BMP-15

Cartilage Oocytes

GDF-5, CDMB-1 GDF-9

McPherron & Lee Human, mouse 1993, Dube et al. 1998

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

24

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 inflammation or rejection. Orthopaedic biomaterials can be implanted into or near a bone fracture to facilitate healing or to compensate for a lack or loss of bone tissue. The materials used in orthopaedic surgery include ceramics, polymers, metals, such as stainless steel, cobaltchromium and titanium and the shape memory alloy NiTi, and resorbable materials, such as bioglass, various modifications of hydroxyapatite and bone grafts. An implant may have bioactive effects on ossification. It may mediate recruitment of mesenchymal cells by growth factors derived from the implant, for example, a bone graft. This is called osteoinduction. In addition, the implant may provide three-dimensional frames for the ingrowth of capillaries and osteoprogenitor cells. In this case, the implant has osteoconductive properties. However, metal alloy implants often give support to bone tissue without any active role in bone formation. The properties of NiTi are here discussed 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 have the ability to return to some previously defined shape or size when subjected to an appropriate stress procedure. The first records of SMA were published about goldcadmium (AuCd) alloy in 1932 (Chang & Read 1951). In 1962, Buehler and co-workers discovered 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 the words: 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 small displacements of atoms occur is called martensitic transformation (MT). This transformation does not change the chemical composition, but results in a new crystal lattice. Thermal cycling, external strain or stress can be used to induce this kind of transformation. Four temperatures characterise temperature-induced transformation. Ms (martensite start temperature) and Mf (martensite finish temperature) are the temperatures during cooling or loading at which transformation from the parent phase into martensite starts and ends. (Fig. 3) The reaction induced by heating or unloading that results in reverse martensitic transformation is characterised by the temperatures As (austenite start temperature) and Af (austenite finish temperature). There is a slight difference between the temperature range of forward and reverse transformations due to the energetically dissimilar transformation pathways. The temperature difference between the phase transformations upon heating and cooling is called hysteresis. In NiTi alloys, it is generally measured as the difference between the temperatures at which 50% of the material is transformed to austenite and 50% to martensite. Finally, the temperature or

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

Fig. 3. Schematic thermal transformation hysteresis loop. T = temperature, %M = martensite percentage, 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 a given strain upon stress release and/or heating. At least three distinctly observable effects are 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 is the strain limit, beyond which strain recovery is incomplete and the material no longer exhibits any memory effect. 2. Two-way (reversible) shape memory is a phenomenon where a martensitic shape is obtained spontaneously during cooling without any external forces. During heating, the original austenite phase is recovered. The characteristic feature of this effect is a lack of hysteresis during temperature cycling. Typically, the magnitude of the twoway shape memory effect is of the order of 1%, and it therefore lacks any real practical interest. 3. Superelasticity is an isothermal phenomenon involving storage of potential energy. Material deformed beyond its apparent yield point fully recovers its initial shape on removal of the load. Significant stress-strain hysteresis is typical of this effect. In near-equiatomic NiTi alloys, the shape memory effect results from a thermoelastic martensitic reaction that occurs as the alloy is cooled through a critical transition temperature range (TTR) (Abujudom et al. 1990). As the alloy is cooled through the TTR, the highly symmetric cubic (B2-type) austenitic () phase transforms through small-scale coordinated atom displacements in a martensitic phase of low crystal symmetry (B19). Factors such as nickel content, aging, thermo-mechanical treatment and addition of alloying elements are important factors in the control of memory behaviour. From the point of view of practical applications, NiTi can exist in three different, temperaturedependent forms: martensite, superelastic and austenite. The martensitic form is ductile, soft and easily deformed. Superelastic NiTi is highly elastic. Lastly, austenitic NiTi is

26 hard, resembling titanium. Commercial NiTi alloys are well used because of their properties. NiTi alloys have large reversible deformations (up to 8 %) at nearly constant stress 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 its problematic production, where a vacuum or an inert atmosphere is needed due to the reactivity of titanium, and the alloy has fewer transformation temperatures than, for example, copper-based alloys. Despite these exceptional physical, chemical and mechanical properties, orthopaedic application of NiTi has been difficult due to the lack of knowledge of the biocompatibility of NiTi on bone.

2.2.2 Cytotoxicity of nickel and titanium

Release of metallic elements from almost all types of alloys has been documented (Brune 1986). The cellular effects of metallic elements do not seem to depend on the type of the cell, which points towards a common mechanism via which cells are affected by metallic compounds (Yamamoto et al. 1998). The cytotoxicity of nickel and titanium has been widely 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 et al. 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 layer on its surface (Hanawa & Ota 1991) Moreover, titanium forms a stable titanium oxide layer on its surface (Kasemo & Lausmaa 1991). This feature is responsible for the good biocompatibility of titanium. Particles derived from titanium implants consist mostly of insoluble titanium oxides or suboxides. They arise from the passivation layer of the implant. If the implant layer is damaged for some reason, the layer is immediately reoxidised. This property protects the alloy and prevents the formation of chemical compounds other than oxides (Hildebrand & Hornez 1998) Titanium is considered one of the best-accepted metals in vitro and in vivo (Doran et al. 1998). However, in vitro Ti4+ ions inhibit osteoclastic activity and reduce osteoblastic protein synthesis (Thompson & Puleo 1996). In a study using the human osteoblastic cell line MG-63, which can be defined as proliferating osteoblasts, titanium was shown to induce IL-6 production (Shida et al. 2000) and, therefore, activate osteoclastogenesis (Kurihara et 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 often neglected, however, that nickel is an essential element for the human body. The dietary exposure 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 the divalent cation receptor (Quarles et al. 1997) or via the Mg2+ channel, which are both situated in plasma membrane. Nickel-containing particles can be phagocytosed by the cell. Phagocytosis of nickel-containing compounds is enhanced by their crystalline nature, negative surface energy, appropriate particle size (2-4 m) and low solubility

27 (Sunderman, Jr. et al. 1987). Ni3S2 and NiO with low in vivo solubility are thought to depend largely upon this pathway (Dunnick et al. 1995). There are two intracellular pathways for nickel. Soluble nickel, such as Ni2+ ions, which enters through receptors or ion channels, binds to cytoplasmic proteins and does not accumulate in the cell nucleus at concentrations 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. This is followed by a decrease of phagocytic intravesicular pH, which releases Ni2+ ions from nickel-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 (Yamamoto et al. 1998). Ni2+ ions increase the proliferation of rabbit bone marrow-derived osteoblasts, but inhibit their maturation and ALP activity and retard mineralisation (Morais et al. 1998). On the contrary, nickel chloride decreased the proliferation of both chondrocytes and fibroblasts (Grant et al. 1994). In addition, nickel inhibits enzymes important for the protection 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 culture models. Human monocytes and microvascular endothelial cells were exposed to pure nickel, titanium, stainless steel and NiTi. The secretion of Il-1 from monocytes and ICAM-1 expression on endothelial cells were measured. NiTi significantly enhanced IL1 secretion by monocytes. This secretion was sufficient to induce ICAM-1 expression on endothelial cells. Somewhat surprisingly, it was found that stainless steel actually released similar quantities of nickel without activating monocytes. Therefore, the release of nickel is not the only reason for the results (Wataha et al. 1999). NiTi was shown to release higher concentrations of Ni2+ in human fibroblast and osteoblast cultures, but this did not seem to have any effect on cell growth (Ryhnen et al. 1997). In vitro studies of minimal essential medium extract cytotoxicity, guinea-pig sensitisation, genotoxicity and salmonella reverse mutation test indicated that NiTi behaves similarly to stainless steel. NiTi did not induce any toxic, allergic or genetic effects (Wever et al. 1997). Genotoxicity of NiTi was also studied in a peripheral blood lymphocyte model with in situ end labeling and electron microscopy. NiTi caused significantly less single-strand breaks on interphase chromatin than pure nickel. Additionally, stainless steel had almost the same effect as nickel. A study of metal ion release in semiphysiological medium revealed 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 have differed. Rondelli et al. showed that NiTi alloys have different corrosion resistance values when 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

28 biocompatibility have been inconsistent. In a study done in simulated physiological solutions, the anodic polarisation behaviours of NiTi and Ti-6Al-4V were compared. NiTi was considered to be biocompatible (Speck & Fraker 1980). However, a surface corrosion study of NiTi and stainless steel under clinical conditions showed low corrosion resistance and, therefore, low biocompatibility for NiTi (Edie et al. 1981). This negative effect was also seen in a comparison of four orthodontic wires in a chloride-induced corrosion test (Sarkar et al. 1983). It is possible that the concentration of nickel affecting cells is higher in vitro than in tissue environments in vivo. Surface characterisation and comparison of NiTi to Ti-6Al4V and stainless steel revealed a TiO2-based layer on both NiTi and Ti-6Al-4V with a low concentration of nickel. Furthermore, a calcium-phosphate layer formed around NiTi, similar to that formed around titanium alloys (Wever et al. 1998). Similar findings were already reported earlier by Hanawa & Ota (1991) and Shabalovskaya & Anderegg (1995). Concentrations of Ni2+ ions in distant organs were not different in rats implanted with NiTi and stainless steel devices as measured from spleen, brain, liver, muscle and kidney 60 weeks after implantation (Ryhnen et al. 1999a). Putters et al. (1992) concluded that NiTi is biocompatible after studying the effects of NiTi, pure nickel and pure titanium in fibroblast cultures. They found nickel to inhibit cell mitosis and NiTi to behave like titanium. Contradictory results were obtained in a study of NiTi, titanium, Co-Cr-Mo, Ti6Al-4V and stainless steel on fibroblasts. NiTi was found to have equally deleterious effects as Co-Cr-Mo on the direct contact of cells to material and was therefore considered 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 capsule surrounding a NiTi implant than were found around other implants composed of Co-Crbased alloys (Castleman et al. 1976). They concluded that this was caused by more abundant metal dissolution from the NiTi implant. On the other hand, a review of in vivo research reports on NiTi covering more than a decade disclosed no allergic reactions, no traces of alloy constituents in the surrounding tissue and no corrosion of implants (Shabalovskaya 1996). Takeshita et al. (1997) studied the response of rat tibiae to NiTi, comparing it to Ti-6Al-4V and stainless steel. They found that the number and area of bone contacts was low around NiTi implants, but the thickness 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 steel screws were implanted for 12 weeks. NiTi had no close contacts, and osteoblasts were disorganised and showed scant osteonectin synthesis. The study, however, had several weaknesses. Firstly, the authors used type III collagen antibody, which is definitively not useful in measuring bone matrix formation. Secondly, the use of antibodies directed against mouse and goat epitopes in rabbit is slightly questionable. Lastly, the sample number (n=6) was too low for statistical analysis (Berger-Gorbet et al. 1996).

29 In a recent study by Fili et al. (2001), NiTi implants were examined. The results disclosed no irritation or adverse reactions in the human body against NiTi-based implants during a study period of 12 months. Their X-ray photoemission spectroscopy (XPS) studies indicated that passivation and reoxidation of NiTi prevents the formation of oxidised nickel particles (Fili et al. 2001). Nickel oxides dissolve easily from the implant and may cause a harmful reaction in the surrounding tissue. NiTi is non-toxic to muscle and perineural tissue, and the fibrous capsule was found to be equally thick around all materials (Ryhnen et al. 1998). NiTi had no negative effect on bone formation, remodelling or consolidation of osteotomies when compared to stainless steel in a 60-week study with intermedullary implantation in rats (Ryhnen et al. 1999a). Normal new bone formation was seen in rats at 26-week follow-up after periosteal implantation (Ryhnen et al. 1999b). NiTi was shown to have similar effects on cell-to-metal adhesion and bone formation at the end of a 26-week follow-up compared to stainless 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 used as biomaterials (Kim & Johnson 1999). The tendency of NiTi to be covered by a titanium oxide layer with only traces of nickel being exposed is considered to be responsible for these good results. However, the good corrosion resistance of NiTi was challenged in a report 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 intermedullary implantation in rats (Ryhnen 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 in orthopaedic applications.

2.2.3 Surface of implant

The major problem associated with the currently used implants is due to inadequate implant-tissue interface properties. The integration of load-bearing implants, such as hip and knee prosthesis and dental implants, into surrounding tissue is important. Both in vivo and in vitro studies have shown that implant surface topography may affect epithelial and connective tissue behaviour. Based on these observations, a mathematical theory of ideal surface pit morphology, dimensions and densities of biomechanical significance was formulated (Hansson & Norton 1999). Another theoretical analysis concluded that if the geometric form of surface roughness is held constant, then the peak elastic stress depends on the form rather than the size of roughness (Skalak & Zhao 2000). However, these mathematical models are frequently used in practice. There are several in vitro studies that show how the physical, chemical and biocompatible properties of implant surface can be improved. Human plasma fibronectin covalently immobilised to NiTi surface improved the attachment of cells (Endo 1995). Coating of the implant surface is not always beneficial. The problem with titaniumbased 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 study which showed that, depending on the treatment, the nickel surface concentration varied between 0.5% and 30%. A hydrogen peroxide-treated NiTi implant was found to be slightly more toxic than pure nickel. Autoclaving in water or steam influenced the NiTi surface 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 calvaria osteoblasts. They tested the effect of these procedures on three different surface roughness areas of pure titanium implants prepared using carbide paper 600-grit surface polishing, sand blasting with diamond paste, or polishing to mirror-like finish. Titanium surfaces treated with plasma glow discharge showed increased osteocalcin expression and ALP activity with increasing surface smoothness. The other methods showed no similar relationship 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 316L stainless steel. All the treatments improved the wear and corrosion resistance of the alloy, but one of them, low-temperature plasma nitriding, impaired fibroblast and osteoblast proliferation and protein synthesis. In another study done using the titanium alloys Ti6Al-4V and Ti-5Al-2.5-Fe, the same authors showed that fibroblasts and osteoblasts grown on untreated or glow-discharged nitrogen-implanted alloys did not show signs of impaired growth. Again, nitriding treatments of the alloys clearly reduced cell proliferation and protein synthesis (Bordji et al. 1996b). When these two studies are taken together, it seems that nitriding treatments of alloys may improve their physical properties but 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 Ti6Al-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 on polystyrene with manually carved grooves, single cells oriented along the long axis of the grooves. 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 a design to compare porous NiTi and coralline hydroxyapatite in rabbit frontal bone implantation. Porous NiTi has an open structure enabling the ingrowth of bone. This allows stable fixation of the implant to bone. The authors claimed that porous NiTi is suitable for craniofacial applications, since no harmful effects were noted and bone ingrowth was good. This study, however, included only 6 rabbits (Simske & Sachdeva 1995). In a recent study on 30 rats, porous titanium mesh coated with Ca-P and filled with allogenic material (rat bone marrow cells) was used to induce ectopic bone formation. Interestingly, control implants without allogenic material did not induce bone formation.

31 Only the implants containing allogenic cells induced ectopic bone formation (Vehof et al. 2000). The biocompatibility of RGD-peptide-coated titanium and gold-coated titanium implants was studied by implanting them in femoral canals of 23 rats. RGD-peptide has a high affinity to integrin 51 and is a common adhesive motif found in proteins throughout the body, including osteopontin, osteonectin and bone sialoprotein. RGDpeptide coating induced the formation of a thicker and more continuous coat of new bone than gold-coated implant (Ferris et al. 1999). Abron et al. (2001) compared a titanium implant of ideal pit density and surface morphology with a non-ideal surface with low pit density and with machined titanium in a rat tibia osteointegration model. The implantation time was 3 weeks and the results showed 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. As literature sited here shows, there are many variations and possibilities to improve tissueimplant interface properties.

3 Aims of the study

New implant materials need to be tested properly before use in patients. Nickel titanium shape memory alloy (NiTi) has unique damping properties, thermal shape memory and superelasticity properties not provided by any other implant alloys. These characteristics make it potentially useful for orthopaedic applications. A lot of interest has been directed towards bone morphogenetic proteins (BMP). There have been several studies of surgical implants with BMP coating or administration. We hypothesised that bone induction could be utilised to improve the biocompatibility of implants. Based on a literature analysis, bone cells tolerate NiTi. We further hypothesised that relevant biological tests could be developed 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 in an 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 was extracted with 1:1 chloroform:methanol mixture for one hour at room temperature with continuous stirring. The particles were decalcified in 0.6 N HCl for 24 hours at +4 C with continuous stirring. Finally, the particles were washed with sterile water by repeating the washing step several times. The particles were lyophilised and stored in sterile vials at -20C. 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, Seinjoki, 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, Synthes GmbH, Switzerland). The surface of stainless steel was electrolytically polished, whereas the surfaces of NiTi and Ti-6Al-4V samples were supplied in a mechanically ground condition. Identical cylindrical implants 1.8 mm in diameter and 6 mm in length were prepared from wire by cutting. The cylindrical implants were filled with decalcified rat bone 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 AISI 316 LVM (12 % Ni, 18 % Cr, 68 % Fe, 2 % Mo; Sandvik, Sweden) ASTM Grade 2 commercially pure titanium (TISTO GmbH; Dsseldorf, Germany) and commercially

34 pure nickel (nickel plating grade used in electrolysis). Metal test discs, 5 mm in diameter and 3 mm thick, were produced from rods with a turning machine. The discs were highly polished 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 different roughness grades produced by mechanical grinding with stone N80 followed by polishing with SiC sandpapers 240, 320, 400, 600, further with sandpapers 800, 1200 and finally with a polishing cloth, the NiTi alloy having an additional treatment with a rubber wheel before polishing. The rubber wheel treatment was done to obtain surface as equal as the titanium 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 cDNAs were amplified in PCR with degenerate oligonucleotide primers derived from the highly conserved carboxy-terminal region of the BMP family to produce cDNAs related to known 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 a known size insert were sequenced with the Perkin Elmer Applied Biosystems BigDye terminator cycle sequencing kit and the ABI PRISM apparatus. Homology comparison analyses were performed with BlastX and BlastN programs, and prediction of protein structure 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 RNA labelling kit (Boehringer Mannheim). The BMP-3b probe was generated from a BMP-3b 0.35 kb fragment in the pGEM-T vector. Tissue sections of 5 m from young reindeer antler were rehydrated with descending concentrations of ethanol, permeabilised with 20 g/ml of RNase-free proteinase K and prehybridised at +42 C with prehybridisation buffer (4 X SSC and 50% formamide). In situ hybridisation was carried out at +55 C for 16 hours in hybridisation buffer (40% formamide, 10 % dextran sulphate, 1% Denharts

35 reagent, 4 X SSC, 10 mM DTT, 1 mg/ml denatured salmon sperm DNA and approximately 0.1 g/ml of labelled antisense RNA probe). Posthybridisation washes were done at 55 C with stringent SSC washes. Hybridised probes were immunologically detected with an antibody to Digoxigenin conjugated to alkaline phosphatase enzyme (Boehringer Mannheim). Controls included hybridisation with the sense (mRNA) probe, RNAse A treatment (20 g/ml) before hybridisation and use of neither antisense nor antidigoxigenin 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 cell cultures or in tissues.

4.3.3.1 DNA laddering

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

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-fixed cells (TACStm TdT kit, R&D Systems, Minneapolis, MN, USA). TdT synthesises fluorescein-labelled dUTP at the 3-OH ends of the broken DNA strands, which are abundant in apoptotic nuclei. To confirm the nuclear morphology, the cells were incubated with the DNA-binding fluorochrome Hoechst 33258 (1mg/ml stock diluted 1:1000 in PBS, Sigma Chemical Co.) for 10 minutes at room temperature. From each disc, six randomly chosen areas (0.849 mm2) were viewed for apoptotic cells under a fluorescence microscope (Nikon Eclipse E600, Nikon, Japan) with a 10 x objective, NA 0.25 (Nikon, Japan).

36

4.4 Cytotoxicity test (III, IV)

The cells on discs were stained with a LIVE/DEADViability/Cytotoxicity kit (Molecular Probes, Eugene, Oregon, USA). The optimal concentration of calcein dye was 1 M and that of ethidium homodimer-1 (EthD-1) dye 0.1 M. The samples were incubated for 15 minutes at 37 C and viewed under a fluorescence microscope. Dead cells (stained red) and live cells (stained green) were counted from six randomly chosen areas (0.849 mm2) on each disc. The cells were counted visually under a fluorescence microscope (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 were seen in each area, while the amount of dead cells per image was calculated as per 1000 cells.

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

The ELISA method (Engvall & Perlmann 1971) is used for the determination of a protein concentration with an immunochemical reaction. TGF-1 and IL-6 cytokine concentrations were detected with the DuoSet human TGF1 and the Duoset rat IL-6 ELISA kits (R&DSystems, Minneapolis, MN, USA). Latent TGF-1 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 (Merck Research Laboratories, West Point, PA, USA). This cell line exhibits features close to those 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 the University of Oulu. All aspects of animal care complied with the Animal Welfare Act and the recommendations of the NIH-PHS Guide for the Care and Use of Laboratory Animals. All the animals used were male Sprague-Dawley rats from the Laboratory Animal Centre, University of Oulu (Oulu, Finland). The age of the animals was 3 months at the beginning of the experiments, and they weighed 350-450 g at that time. During the experiment, the rats were housed in groups of 3-6 in Macrolon IV polycarbonate cages in a 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 and implants were immediately removed and stored using the formalin fixation procedure.

4.8 Ectopic bone formation assay

All rats were anaesthetised with a fentanylcitrate (80 g/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 was shaven around the implantation site and the skin was sterilised by brushing it with chlorhexidin before the operation. Study I: Decalcified matrix made of one-month-old, four-month-old and cast reindeer antlers and rat long bones (10 of each) was implanted, with two implants per rat. A single skin incision of about 2 cm was made in midline between the scapulae. The implants were placed in gelatin capsules under the fascia of the right (allogenic implant or 4-month-old antler) and left (cast antler or 1-month-old antler) latissimus dorsi muscle. After 3 weeks the 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 (only allogenic and cast antler matrix, 10 rats/sample). Study II: Ten NiTi, 8 stainless steel, and 8 Ti-6Al-4V implants with rat decalcified bone matrix packaged into gelatin capsules were implanted. All three implant materials were placed under the fascia of the latissimus dorsi muscles of each rat (n=10). Ten control rats received gelatin capsules containing allogenic matrix without any metal implant through a similar surgical operation. The animals were euthanised and the formed ossicles 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 m sections (I) or 25 m sections (II) were stained with the von Kossa and Masson-Goldner-Trichrome methods for histology and morphometric 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 (XCT 920A, Norland Stratec Medizintechnik GmbH, Birkenfeld, Germany). Pixel size was 0.145 m2 and section thickness 1.25 mm. In study II, pQCT scans were taken 1mm apart from the implant based on a scout view image.

38

4.9.2 Light microscopy (I, II)

The morphology and histology of the von Kossa and the Masson-Goldner-Trichrome stained 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 (Sony XCT930P, Sony, Japan), a microscope (Nikon Optiphot II, Nikon, Japan) and a personal computer with a digitiser (Matrox Image 640 with CLD color board, Imaging Technology, USA). Study I: The proportional area of mineralisation in each implant was measured. Von Kossa stained sections were analysed for black staining of mineralised tissue, and the area of mineralisation was compared to the ossicle area. Study II: Polarised light microscopy was used to distinguish between fibrotic tissue and bone. The proportional areas of fibrotic tissue and non-resorbed bone matrix powder were compared to the implant area. The target area was cropped by excluding the new woven bone, but including fibrotic tissue and non-resorbed initial rat bone matrix. The proportional new bone area versus ossicle area and the proportional area of cartilage versus ossicle area were also examined. Study III, IV: The number of focal contacts was measured with a digital image analyser (MCID M4 v.3.0.re.1.1, Imaging Research Inc., Ontario, Canada). The examined region of interest was 20117 m2. Confocal microscope images were semiautomatically segmented on red color intensity, hue and saturation. The interactively defined focal contacts 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-crypton laser (Omnichrome, Chino, CA, USA), a Leitz Aristoplan microscope, and software version 1.05 (Leica Lasertechnik GmbH, Heidelberg, Germany). Rat osteosarcoma cells were cultured in MEM-FCS growth medium on different alloys for 48 hours, fixed with 4 % PFA for 10 minutes at room temperature and permeabilised with 0.1 % Triton-X-100 in PBS for 10 minutes on ice. For immunofluoresence staining, the samples were incubated with primary monoclonal paxillin antibody (ZYMED Laboratories, Inc., San Francisco, CA, USA) for 45 minutes on ice. The samples were rinsed thoroughly several times with PBS before the staining was completed with rhodamine-conjugated rabbit anti-mouse immunoglobulin secondary antibodies (DAKO, Glostrup, Denmark) for 30 minutes 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 Chemical Co.) for 10 minutes at room temperature. The focal contacts were studied under a confocal microscope LSM 510 equipped with an inverted microscope Axiovert 100M and a 63 x (NA 1.2/w) water immersion objective (Zeiss, Gttingen, Germany). From each sample disc, 6 frames were scanned with 1024 x 1024 frame size (pixel size 0.81 m2).

4.9.5 Atomic force microscopy (AFM) (IV)

Atomic force microscopy was used to determine the surface roughness parameters of the NiTi and titanium alloys with different surface characteristics. AFM measurements were performed with an Explorer system (Thermomicroscopes, Sunnyvale, CA, USA) and SPMLabNT 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 the cantilever. This motion of the cantilever as it applies force to the sample, was used in a feedback loop to control the Z piezo, and the constant cantilever deflection was maintained. The scanning parameters were adjusted according to the surface topography. The size of the scanned area was100 x 100 m and the resolution of image was 400 x 400 pixel.

4.9.6 Statistical analysis (I-V)

Mean values and standard deviations were computed. Analysis of variance (ANOVA) and Students t-test were utilised to assess the level of significance of the differences between the experimental groups. All statistical analyses were performed with commercial software (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 first primers were designed from predicted homologues of the BMPs mature region cysteines numbered 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 potential glycolysation site in the precursor region of the BMP-3b and from the sequenced reindeer mature peptide region cysteines 4-5. PCR with these primers produced 1050 base pairs of the BMP-3b sequence. 5RACE produced some more nucleotides, but the starting codon and the 5 untranslated region were not solved. We were able to produce 322 nucleotides of the 3 untranslated region. Overall, the produced cDNA sequence contained 1324 nucleotides 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 the mature region and 86 % in the precursor region. The amino acid level homologies were 90 % and 84 %, respectively. The partial coding sequence of reindeer BMP-3b was deposited in the GenBank under accession number AF300813.

41

5.1.3 In situ hybridisation

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

5.1.4 Ectopic bone formation assay of antler matrix

Ectopic bone formation assay was used to study the bone induction potential of different agents in live animals. We compared allogenic bone matrix with different stages of antler maturation. None of the test animals showed any signs of inflammation when assessed visually. Round ossicles were already seen in the 3-week implantation groups, but the 8week cast antler ossicles showed irregular shapes and were softer than the same matrix in the 3-week implantation. Histological evaluation of samples stained with von Kossa and toluidene blue counterstain revealed differences in the ossicle morphologies of the different matrix preparations. The ossicles of the allogenic matrix showed the phenotype of endochondral ossification, but the antler matrix-induced ossicles expressed mesenchymal condensation similar to intramembranous ossification. Mineralising nodules were seen around the capillaries in cast antler ossicles. The implanted antler matrix 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 the allogenic and antler matrices were compared. In addition, the ossicles induced by cast antler matrix had significantly higher mineral density than the other two antler matrixinduced ossicles. When a longer implantation period was used, the allogenic matrix had high BMD, while the 8-week ossicles of antler matrix did not have adequate mineralisation for x-ray attenuation to be measured with pQCT. The histological von Kossa staining of the accumulated mineral was in line with the BMD. The proportional mineralised area in the implants of the allogenic matrix at 3 weeks was 3.72.1 %. The proportional mineralised area was significantly smaller (p<0.005) in all antler matrix implants. When the four-month (0.250.17 %) and onemonth (0.080.05 %) antler matrix implants were compared to the cast antler matrix at 3 weeks (0.650.21 %), 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 (1.20.01 %) compared to the allogenic matrix (11.60.1 %, p<0.01). The measurement of the proportional amount of cartilage showed that there was no normal cartilage in any of the antler matrix induced ossicles. Instead, the original matrix implant material with membranotic ossification, mesenchymal cell condensations and calcified nodules were observed. In the 3-week allogenic implant, the proportion of cartilage was 2.40.2 % of the total area. The above observations of antler studies are shown summarised in table 2.

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

Qualifiers BMD Mineralization area Endochondral ossification Allogenic Dense Large Evident Cast antler Intermediate Small Not detected 4-month antler Sparse Very small Not detected 1-month antler Very sparse Very small Not detected

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 bone formation. After 8 weeks, advanced endochondral bone formation was evident. Most of the ossicles were filled with new woven bone, and there were very few decalcified bone particles left. A very close contact between the implant and the new woven bone without fibrotic material was observed in some areas of two Stst samples, two Ti-6Al-4V samples and one NiTi sample. BMD in the control group was 35069 mg/cm3. The values of NiTi, Stst and Ti-6Al-4V were 36087 mg/cm3, 28543 mg/cm3 and 29373 mg/cm3, respectively. Histomorphometric studies showed some differences between the tested materials in view of bone induction efficacy. The mean proportion of fibrotic tissue and bone matrix powder was higher in the Ti-6Al-4V than in the other two alloys tested. The proportion of cartilage was higher in the NiTi group (0.160.09) than in the Ti-6Al-4V group (0.050.04) (p<0.05). The proportion of cartilage in the control group was low. The amount of new bone, measured as an area proportional to the area of the ossicle was high in the control group. Instead, the Stst group had significantly less new bone compared to the 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 BMD Fibrotic tissue area Cartilage area Bone area Allogenic Dense Not detected Little Large area NiTi Dense Intermediate Large area Intermediate Ti-6Al-4V Intermediate Large area Very little Intermediate Stainless steel Intermediate Intermediate Intermediate Little

43

5.2.2 Surface roughness (IV)

The surface roughness of materials in study IV was assessed with AFM. The average roughness (Ra) values are expressed in table 4. Table 4. Average surface roughness of implant materials (nm) mean 1 SD.
Materials NiTi TiI [Ti-6Al-2.2Mo1.3Cr(850 C)] TiII [Ti-6Al-2.2Mo1.3Cr(1050 C)] 1200 9541 304310 33939 600 15520 1428173 43610 80 362209 147938 73249

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

5.2.3 Cell viability (III, IV)

During the 48-hour culture period, cells reached confluency in all materials with a smooth surface. Cells cultured on NiTi survived, as did those on titanium. The cell death rate 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 seemed larger than those on smooth surfaces. All cultures, except TiII80 group, reached complete confluency in 48 hours. The cytotoxicity test showed cell viability to be significantly better for the roughest NiTi and TiI surfaces than the other surfaces in the test groups. The cell 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 compositions and surface roughness values. Mean 1 SD.
Materials NiTi TiI [Ti-6Al-2.2Mo1.3Cr(850 C)] TiII [Ti-6Al-2.2Mo1.3Cr(1050 C)] Pure nickel Pure titanium Stainless steel 30.820.9 2.93.3 12.717.5 Smooth 2.43.6 1200 2226 26.626.7 25.143.3 600 23.127.9 33.637 22.828.3 80 11.921 14.821.3 33.529

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

44
Stst Ti Ni NiTi NiTi1200 NiTi600 NiTi80 TiI1200 TiI600 TiI80 TiII1200 TiII600 TiII80

80 70 Dead cells/1000 cells 60 50 40 30 20 10 0

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, we stained the cells with TUNEL assay. The results of the TUNEL assay were combined with the number of dead cells detected in the viability assay, and the percentage of apoptotic cells out of all dead cells was calculated (Table 6). Table 6. Ratio of apoptotic cells in cultures on different materials. Mean 1 SD.
Materials NiTi Titanium Stainless steel Nickel *: p<0.05 to titanium, ***:p<0.001 Apoptotic cells/1000 cells 1.932.1 2.981.97 1.1.0.96 0.620.65* % apoptotic cells/dead cells 46 62 5.3*** 1.2***

5.2.5 Cell attachment (III, IV)

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

45 significant between the Ni group and the Ti group. NiTi 80 strongly stimulated the formation 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 surface appeared 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.
Materials NiTi TiI [Ti-6Al-2.2Mo1.3Cr(850 C)] TiII [Ti-6Al-2.2Mo1.3Cr(1050 C)] Pure Ti Pure Ni Stainless steel 462.5362 261.5226 335.8239 Smooth 744.6427 1200 485343 657.6355 350194 600 460272 423.4222 246.8156 80 611325 269177 223151

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

1400.0 1200.0 Focal adhesions/frame 1000.0 800.0 600.0 400.0 200.0 0.0

Stst Ni Ti NiTi NiTi1200 NiTi600 NiTi80 TiI1200 TiI600 TiI80 TiII1200 TiII600 TiII80

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

46

5.2.6 Detection of TGF-1 and IL-6 cytokines (V)

To find out how growth factor production might change on different materials, we measured TGF-1 and IL-6 concentrations with the ELISA method. There was quite a lot of intragroup variation, but overall, the rough NiTi surface showed the highest concentrations of TGF-1. In all sample groups, the IL-6 concentrations were too low to be detected with the system used. Table 8 and figure 6 show the results of the TGF-1 ELISA measurements. Table 8. TGF-1 concentrations (ng/ml) in various culture media. Mean 1 SD.
Materials NiTi TiI [Ti-6Al-2.2Mo1.3Cr(850 C)] TiII [Ti-6Al-2.2Mo1.3Cr(1050 C)] Pure Ti Pure Ni Stainless steel 1.210.22 1.060.42 1.250.21 Smooth 1.290.32 1200 1.160.55 0.860.19 1.370.18 600 1.20.69 1.070.38 1.270.25 80 1.480.17 0.960.6 1.50.17

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

2.5

Stst Ti Ni NiTi NiTi1200 NiTi600 NiTi80 TiI1200 TiI600 TiI80 TiII1200 TiII600 TiII80

2 TGF-beta1 ng/ml

1.5

0.5

Fig. 6. TGF-1 concentrations of cell cultures on different materials.

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 used degenerate oligonucleotide primers in the amplification of a cDNA pool generated from 1-month antler. Degenerate PCR has been used before in the detection of several mammalian bone morphogenetic proteins (Chang et al. 1994). This method enables the discovery 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 nucleotides coding for 441 amino acids. Previously, BMP-2 and -4 cDNAs have been cloned from antler of red deer (Feng et al. 1995). These cDNAs prove that antlers express several different messenger RNAs of the BMP family, which probably have roles in antlerogenesis. Unfortunately, we did not succeed in the amplification and sequencing of the 5 end of BMP-3b cDNA. Very often, the 5 ends of the coding sequences are rich in guanine and cytosine nucleotides. These nucleotides form three hydrogen bonds with each other, which produces stable secondary structures. The expected size of the coding sequence 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 BMP3b 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 BMP family members. In a recent study, BMP-3 was found to inhibit the effects on BMP-2 responsiveness on osteoblastic cell lines. The explanation for this may be that BMP-3

48 signals through the TGF-/activin pathway and thereby antagonise BMP-2 signalling. Homozygotic knockout mice, which lack BMP-3, survive without any skeletal changes observed in embryos or neonates. In adult mice, the lack of BMP-3 doubles the trabecular bone mass compared to wild-type mice (Bahamonde & Lyons 2001). On the contrary, BMP-3b knockout mice have no phenotypic changes, probably due to compensation by other 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 rat calvarial osteoblasts (Hino et al. 1999). To study BMP-3b in antlerogenesis, we studied its localization in antlers using in situ hybridisation. This method revealed BMP-3b expression mostly in mature cells in the antler centre near the capillaries. Unfortunately, we were unable to produce slices of calcified antler tissue. The study of more mature antlers would have given new information about BMP-3b expression. In addition, it would have been most useful to detect changes in BMP-3b expression over time by using in situ hybridisation at different stages of antler maturation. The role of BMP-3b in reindeer antler development remains to be solved, but this growth 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 the effect of different compounds on the induction of bone. Our approach to use material packaged in gelatin capsules makes the handling of the test materials easier. The use of the back of the animal as the implantation site causes minimal harm to the animal. The ectopic bone formation assay showed that decalcified antler matrix contains agents that activate 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 extensively before its use. The rationale of using immunogenic matrix, such as reindeer antler, for osteoinduction is that the antler is a rich source of bone growth factors, possibly even some currently unknown members of the BMP family. Even though a large number of BMPs have already been discovered, the most effective ones might not yet been found. It is quite clear that growth factors to be used in clinical applications will be produced with recombinant DNA technology, not by extraction from native sources. Agents can be engineered so that they have reduced immunological effects. The production of fully humanized proteins will reduce the need for immunosuppressive medication, which is needed when allografts are used.

49

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 endochondral bone formation. Since the allogenic bone matrix induces endochondral ossification, the method can be used to monitor the effects of materials embedded into the allogenic matrix on bone formation. We refined the method of ectopic bone formation assay to study the biocompatibility of NiTi. Implantation of NiTi together with rat bone matrix enabled us to monitor the effect of the alloy on bone formation. Ectopic bone formation assay of reindeer antler had disclosed that the allogenic matrix forms fully mineralised ossicles 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, namely stainless steel and Ti-6Al-4V. This comparison showed that these alloys behave in a very similar manner. Only the area of cartilage was higher in NiTi ossicles compared to other metal alloys. This may indicate that the formation of cartilage is faster in ossicles formed in the presence of NiTi than the other alloys studied. Since the control ossicles were almost fully mineralized, they were expected to have a low cartilage content and high mineral density. To our surprise, NiTi ossicles showed mineral density equal to the control ossicles, even though the bone area was smaller than in the controls. NiTi was closer to Ti-6Al-4V than to stainless steel as far as bone mineral density and bone area were concerned, but fibrotic tissue was scant compared to the Ti-6Al-4V group. On the other hand, these results might imply that bone formation and mineralization are more effective in Ti-6Al-4V ossicles, which leaves only a minor role for cartilage between the resorption 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 for osteoblasts because of their well-differentiated osteoblast phenotype and their ability to undergo apoptosis (Ihbe et al. 1998). In long-term implantation, mineralisation of bone is an important factor for the success of implantation. The use of ROS-17/2.8 cells enabled us to monitor the effects of the implant material on the differentiation of osteoblastic cells. A relatively long culture time, 48 hours, was chosen to ensure nickel release. After 48 hours, the release of nickel decreases rapidly, probably due to the titanium oxide layer that is formed on the surface of the NiTi implant (Ryhnen et al. 1997). Cultures were performed using low cell numbers (5000 cells/disc) to keep the cells as a monolayer during the 48-hour culture period. High cell densities reduce the sensitivity of cells to metal ions (Wataha et al. 1993). The number of viable cells was counted using fluorescence microscopy. Flow cytometry could also have been used, but it would have necessitated trypsinisation of cells to detach them from the implant surface. Due to the low cell number and the loss of cells during the handling of the samples, this would

50 probably have given too low cell yields. We calculated the number of both dead and live cells to compare their ratio. The high number of dead cells on nickel samples was no surprise, since nickel is known to be toxic. On the contrary, NiTi showed a low ratio of dead to live cells, as did pure titanium. We believe that this shows the good biocompatibility of NiTi alloy, especially because stainless steel had a slightly worse ratio. The other methods frequently used to assess the viability of osteoblasts are based on DNA synthesis with thymidine incorporation, the MTT test, and ALP expression. The MTT test is based on the reduction of diphenyltetrazolium bromide salt by mitochondrial enzymes. Frequently, ALP activity is measured using a test in which the enzyme hydrolyses p-nitrophenylphosphate. Alternatively, ALP can be stained histochemically. The measurement of ALP activity is considered to be a more sensitive method for the detection 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 and TiI surfaces were better tolerated than the other surfaces. The better results obtained with NiTi than the titanium alloy are probably due to nickel being the only released particle from 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 cell viability, possibly because of the heat treatment temperature. The rank order of cell viability 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 many possibilities to detect apoptosis. These methods are based either on the demonstration of nuclear morphology or on the measurement of the activity of certain cytoplasmic enzymes, caspases, which are needed in cell lysis. Detection of apoptosis is widely used in biocompatibility studies of biomaterials. Our studies showed very low rates of apoptosis induced by the alloys studied, even though NiTi and titanium had higher apoptotic indices than pure nickel and stainless steel. ROS17/2.8 cell cultures have been shown to have a lag phase of growth until 72 hours (Ihbe et al. 1998). This may be the reason for the low apoptosis in our study: because cultures would have still been at the lag phase of growth. In addition, it probably explains why no signs of apoptosis were seen using DNA laddering. If the cells were still in their of exponential growth phase, with a very low apoptosis rate, the detection limit (20 % apoptotic cells) of the DNA laddering technique was probably not reached. Cell viability was similar on rough and smooth surfaces. Therefore, in the study on the effect of surface roughness on cell viability, TUNEL assay was not used to measure apoptosis. The single problem in the detection of apoptosis is that there are many phases in apoptosis. One assay does not necessarily cover every apoptotic event. Because of this, it is possible that apoptotic cells, which are in different phases, are not all detected in one assay. We tried to avoid these problems by staining the TUNEL-stained cells with a nuclear stain and thereby to double-check the nuclear morphology.

51 We found it informative to calculate the ratio of apoptotic to dead cells. This parameter is useful for the determination of the way cells die on implant materials. Our results showed 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 to be important for good osteointegration. The focal adhesions are artificial attachment sites of 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 forms the cell membrane spanning compartment and several other proteins inside the cell respond to cell motility signals (Gumbiner 1993). We studied the attachment of ROS-17/2.8 cells on different alloys with different surface roughness values. To analyse the effect of the implant on cell attachment, focal adhesions of the cells were stained with paxillin antibody. The number of focal adhesions per picture frame could be used as a measure of cell adhesion because the cultures were confluent at the time of assay. Another measure of adhesion would have been the number of 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 the appearance of the cells; only the cells cultured on nickel had some morphological changes. In our study, the cells cultured on NiTi had almost twice as many focal contacts as those cultured on pure titanium. This was an interesting result, since the morphology of the cells and the measures of cell viability showed marked similarities between titanium and NiTi. Hunter et al. studied osteoblast and fibroblast attachment on different orthopaedic biomaterials and found no significant differences in the attachment of osteoblasts to different materials (Hunter et al. 1995). Their scanning electron microscope (SEM) observations were in line with morphometric data: cells with high numbers of focal adhesions were well spread and flattened, while cells with a low number of focal adhesions were rounded and less spread. In our study, cultures on pure nickel had the lowest numbers of focal adhesions, and these cells were round. We conclude that cell attachment to NiTi surfaces is good and definitely better than attachment to pure nickel surfaces. The roughness of the surface affected cell attachment in such a way that the cells oriented along the grooves on all of the three implant materials. This observation demonstrates that ROS-17/2.8 cells exhibit features common to rat bone marrow cells and fibroblasts, 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 surface roughness values. The attachment sites of cells on NiTi were numerous in all surface roughness groups. Titanium alloys with two different heat treatments showed distinct differences in the cytocompatibility of rough surfaces. There were significantly fewer focal adhesions in the cells cultured on material heated at 1050 C than on material heated at 850 C. The heat treatment may stress material surface and, consequenctly, decrease

52 the ion release from the surface in culture. These ions and the compounds formed of them in the cells could be cytotoxic. Unfortunately, we do not have data about the chemical surface characteristics of these differently treated titanium alloys. However, it seems that osteoblasts sense implant surface characteristics and that this affects their attachment. There are no reports on the effect of austenitic versus martensitic surface on cytocompatibility. As a conclusion, the materials studied in the present work can be placed in the following rank order as to cell attachment: TiII<TiI<NiTi. This order is the same as was observed in the cell viability assays.

6.2.5 Detection of TGF-1 and IL-6 cytokines (V)

ELISA measurements of TGF-1 showed high intragroup variance. However, different patterns in TGF-1 concentration where induced by different materials. TGF-1 concentrations increased in proportion to the roughness of NiTi and peaked in the middle roughness group of the TiI implants, but reached the minimum value in the middle roughness group of the TiII samples. TGF-1 is a potent mitogen for osteoblastic cells (Robey et al. 1987). Because TGF-1 was produced in all cultures, except those on nickel and TiI surfaces, we conclude that these cells signalled of proliferation. In contrast to viability and attachment, TGF-1 concentrations lead to the following ranking: TiI<TiIINiTi. Overall, rough and smooth NiTi implants were cytocompatible. One reason for the good results obtained with the use of smooth NiTi implants might be that the smooth implants were prepared of austenite alloy. All the ranking orders used are shown in table 9. Osteoblasts are cells that are located close to orthopaedic implants. Remodelling starts when the cells close to the implant signal to osteoclasts to start resorption. IL-6 could perhaps 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 from the 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 would explain the lack of IL-6, since other studies have shown that the cytokine response is rapid in cell cultures (Shida et al. 2000). Table 9. Ranking orders of NiTi, TiI and TiII found in this study.
Property Cell viability Cell attachment TGF-1 production Ranking order TiII<TiI<NiTi TiII<TiI<NiTi TiI<TiIINiti

7 Conclusions
The degenerate oligonucleotide primer PCR produced a BMP sequence with high homology to human and rat BMP-3b. This cDNA was named reindeer BMP-3b. The eventual osteoinductive properties of reindeer antler matrix were tested using a modified ectopic bone formation assay. It was shown that decalcified reindeer antler matrix was immunogenic and induced slight mineralization. Reindeer antler is perhaps a rich source of various BMPs, but the matrix must be more purified in vivo. However, if the reindeer antler matrix had had clear osteoinductive properties, the combined use of matrix and NiTi might have been possible. We successfully modified the ectopic bone formation assay, so that it could be used for a biocompatibility study of NiTi. The modification used is animal-friendly and easy to perform. This method can be used for screening of biomaterials for their effects on bone formation prior to clinical use in animals or humans. Ossicles associated with NiTi implants had similar characteristics as those associated with Ti-6Al-4V and stainless steel implants. Because no inflammatory reactions were observed in the ossicles and histomorphometric analysis showed endochondral bone formation, NiTi can be considered to be biocompatible. Compared with pure titanium, pure nickel and stainless steel, NiTi was a well-tolerated osteoblast culture surface. NiTi showed cell viability similar to that of pure titanium. Cell viability on stainless steel came close to that of nickel. The high number of focal contacts showed that cells attached well to NiTi surfaces. Cells had fewer focal contacts even when cultured on pure titanium. Surface roughness affected the viability, focal contacts and orientation of ROS-17/2.8 cells. Surface roughness was sandpaper-modified of three different alloys: NiTi and Ti6Al-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 with heat treatment at +850 C induced good cell attachment. Less rough NiTi and titanium alloy surfaces with heat treatment at +1050 C showed slightly higher cell death rates and fewer focal contacts. Cells grown on the rough surfaces oriented along the long axis of the grinding grooves.

54 Cytokine detection in the supernatant of cells cultured on alloys turned out to be somewhat problematic as to IL-6, but not as to TGF-1, due to the low cell counts. We conclude that the ELISA method used in this study can be used to screen cytokine signalling of osteoblasts cultured on biomaterials.

8 References
Abbracchio MP, Simmons-Hansen J & Costa M (1982a) Cytoplasmic dissolution of phagocytized crystalline nickel sulfide particles: a prerequisite for nuclear uptake of nickel. J Toxicol Environ Health 9:663-676. Abbracchio MP, Heck JD & Costa M (1982b) The phagocytosis and transforming activity of crystalline metal sulfide particles are related to their negative surface charge. Carcinogenesis 3:175-180. Abercrombie M & Ambrose EJ (1958) Interference microscope studies of cell contacts in tissue culture. Exp Cell Res 15:332-345. Abron A, Hopfensperger M, Thompson J & Cooper LF (2001) Evaluation of a predictive model for implant surface topography effects on early osseointegration in the rat tibia model. J Prosthet Dent 85:40-46. Abujudom DN, Thoma PE, & Fariabi S (1990) The effect of cold work and heat treatment on the phase transformations of near equiatomic NiTi shape memory alloy. Mater Sci Forum 56-58:565570. Albrektsson T, Brnemark PI, Hansson HA & Lindstrm J (1981) Osseointegrated titanium implants. Requirements for ensuring a long-lasting, direct bone-to-implant anchorage in man. Acta Orthop Scand 52:155-170. Altschul SF, Gish W, Miller W, Myers EW & Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403-410. Anderson HC (1969) Vesicles associated with calcification in the matrix of epiphyseal cartilage. J Cell Biol 41:59-72. Assad M, Lemieux N, Rivard CH & Yahia LH (1999) Comparative in vitro biocompatibility of nickel-titanium, pure nickel, pure titanium, and stainless steel: genotoxicity and atomic absorption evalution. Biomed Mat Eng 9:1-12. Assad M, Lombardi S, Berneche S, Desrosiers EA, Yahia LH & Rivard CH (1994) Assays of cytotoxicity of the Nickel-Titanium shape memory alloy. Ann Chir 48:731-736. Bahamonde ME & Lyons KM (2001) BMP3: to be or not to be a BMP. J Bone Joint Surg 83:S56-S62. Banks WJ (1974) The ossification process of the developing antler of the white tailed deer (Odocoileus virginianus). Calcif Tissue Res 14:257-257. Berger-Gorbet M, Broxup B, Rivard C & Yahia LH (1996) Biocompatibility testing of NiTi screws using immunohistochemistry on sections containing metallic implants. J Biomed Mat Res 32:243248. Bilbe G, Roberts E, Birch M & Evans DB (1996) PCR phenotyping of cytokines, growth factors and their receptors and bone matrix proteins in human osteoblast-like cell lines. Bone 19:437-445.

56
Bordji K, Jouzeau JY, Mainard D, Payan E, Delagoutte JP & Netter P (1996a) Evaluation of the effect of three surface treatments on the biocompatibility of 316L stainless steel using human differentiated cells. Biomaterials 17:491-500. Bordji K, Jouzeau JY, Mainard D, Payan E, Netter P, Rie KT, Stucky T & Hage-Ali M (1996b) Cytocompatibility of Ti-6Al-4V and Ti-5Al-2.5Fe alloys according to three surface treatments, using human fibroblasts and osteoblasts. Biomaterials 17:929-940. Bostrm M, Lane J, Berberian W, Missri A, Tomin E, Weiland A, Doty S, Glaser D & Rosen V (1995) Immunolocalization and expression of bone morphogenetic proteins 2 ans 4 in fracture healing. J Orthop Rel Res 13:357-367. Brnemark PI, Adell R, Breine U, Hansson BO, Lindstrm J & Ohlsson A (1969) Intra-osseous anchorage of dental prostheses. I. Experimental studies. Scand J Plast Reconstr Surg 3:81-100. Brighton CT & Hunt RM (1991) Early histological and ultrastructural changes in medullary fracture callus. J Bone Joint Surg 73:832-847. Bruinink A & Wintermantel E (2001) Grooves affect primary bone marrow but not osteoblastic MC3T3-E1 cell cultures. Biomaterials 22:2465-2473. Brune D (1986) Metal release from dental biomaterials. Biomaterials 7:163-175. Buehler WJ & Wang FE (1968) A summary of recent research on the Nitinol* alloys and their potential application in ocean engineering. Ocean Engineering 1:105-120. Cartana J, Arola L & Romeu A (1989) Characterization of the inhibition effect induced by nickel on glucose-6-phosphate dehydrogenase and glutathione reductase. Enzyme 41:1-5. Castleman LS, Motzkin SM, Alicandri FP & Bonawit VL (1976) Biocompatibility of nitinol alloy as an implant material. J Biomed Mat Res 10:695-731. Celeste AJ, Iannazzi JA, Taylor RC, Hewick RM, Rosen V, Wang EA & Wozney JM (1990) Identification of transforming growth factor beta family members present in bone-inductive protein purified from bovine bone. Proc Natl Acad Sci U S A 87:9843-9847. Celeste AJ, Ross JL, Yamali N, & Wozney JM (1995) The molecular cloning of human bone morphogenetic proteins-10, -11, and -12, three new members of the transforming growth factor-beta superfamily. J Bone Miner Res 10:S336. Chang LC & Read TA (1951) Trans AIME 191:47. Chang SC, Hoang B, Thomas JT, Vukicevic S, Luyten FP et al. (1994) Cartilage-derived morphogenetic proteins. New members of the transforming growth factor-beta superfamily predominantly expressed in long bones during human embryonic development. J Biol Chem 269:28227-28234. Chapman D.I. (1975) Antlers - bone of contention. Mammal Rev 5:121-172. Chow JW, Wilson AJ, Chambers TJ & Fox SW (1998) Mechanical loading stimulates bone formation by reactivation of bone lining cells in 13-week-old rats. J Bone Miner Res 13:1760-1767. Cook SD, Dalton JE, Tan EH, Whitecloud TS III & Rueger DC (1994a) In vivo evaluation of recombinant human osteogenic protein (rhOP-1) implants as a bone graft substitute for spinal fusions. Spine 19:1655-1663. Cook SD, Baffes GC, Wolfe MW, Sampath TK, Rueger DC & Whitecloud TS III (1994b) The effect of recombinant human osteogenic protein-1 on healing of large segmental bone defects. J Bone Joint Surg 76:827-838. Cook SD & Rueger DC (1996) Osteogenic protein-1: biology and applications. Clin Orthop 324:2938. Cunningham NS, Jenkins NA, Gilbert DJ, Copeland NG, Reddi AH & Lee SJ (1995) Growth/differentiation factor-10: a new member of the transforming growth factor-beta superfamily related to bone morphogenetic protein-3. Growth Factors 12:99-109. Dean DD, Schwartz Z, Bonewald L, Muniz OE, Morales S et al. (1994) Matrix vesicles produced by osteoblast-like cells in culture become significantly enriched in proteoglycan-degrading metalloproteinases after addition of beta-glycerophosphate and ascorbic acid. Calcif Tissue Int 54:399408.

57
Doran A, Law FC, Allen MJ & Rushton N (1998) Neoplastic transformation of cells by soluble but not particulate forms of metals used in orthopaedic implants. Biomaterials 19:751-759. Dube JL & Celeste AJ (1995) Human bone morphogenetic protein-13, a molecule which is highly related to human bone morphogenetic-12 protein. J Bone Miner Res 10:S336. Dube JL, Wang P, Elvin J, Lyons KM, Celeste AJ & Matzuk MM (1998) The bone morphogenetic protein 15 gene is X-linked and expressed in oocytes. Mol Endocrinol 12:1809-1817. Dunnick JK, Elwell MR, Radovsky AE, Benson JM, et al. (1995) Comparative carcinogenic effects of nickel subsulfide, nickel oxide, or nickel sulfate hexahydrate chronic exposures in the lung. Cancer Res 55:5251-5256. Ducy P, Zhang R, Geoffroy V, Ridall AL & Karsenty G (1997) Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89:747-754. Edie JW, Andreasen GF & Zaytoun MP (1981) Surface corrosion of nitinol and stainless steel under clinical conditions. Angle Orthod 51:319-324. Einhorn TA, Majeska RJ, Rush EB, & et al. (1995) The expression of cytokine activity by fracture callus. J Bone Miner Res 10:1272-1281. Eisenbarth E, Meyle J, Nachtigall W & Breme J (1996) Influence of the surface structure of titanium materials on the adhesion of fibroblasts. Biomaterials 17:1399-1403. Endo K (1995) Chemical modification of metallic implant surfaces with biofunctional proteins (Part 1). Molecular structure and biological activity of a modified NiTi alloy surface. Dent Mater J 14:185-198. Engvall E & Perlman P (1971) Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8:871-874. Feng JQ, Chen D, Esparza J, Harris MA, Mundy GR & Harris SE (1995) Deer antler tissue contains two types of bone morphogenetic protein 4 mRNA transcripts. Biochem Biophys Acta 1263:163168. Feng JQ, Chen D, Ghoshchoudhury N, Esparza J, Mundy GR & Harris SE (1997) Bone morphogenetic protein 2 transcripts in rapidly developing deer antler tissue contain an extended 5' non -coding region arising from a distal promoter. Biochem Biophys Acta 1350:47-52. Feng JQ, Harris MA, Ghosh-Choudhury N, Feng M, Mundy GR & Harris SE (1994) Structure and sequence of mouse bone morphogenetic protein-2 gene (BMP-2): comparison of the structures and promoter regions of BMP-2 and BMP-4 genes. Biochim Biophys Acta 1218:221-224. Ferris DM, Moodie GD, Dimond PM, Gioranni CW, Ehrlich MG & Valentini RF (1999) RGD-coated titanium implants stimulate increased bone formation in vivo. Biomaterials 20:2323-2331. Fili P, Lausmaa J, Musialek J & Mazanec K (2001) Structure and surface of TiNi human implants. Biomaterials 22:2131-2138. Friedlaender GE, Clayton RP, Cole JD, Cook SD et al. (2001) Osteogenic protein-1 (Bone morphogenetic protein-7) in the treatment of tibial nonunions. J Bone Joint Surg (Am) 83:S151-S158. Gavrieli Y, Sherman Y & Ben Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493-501. Gegenbaur C (1864) Uber die Bildung des Knochengewebes. Jena Zeitschrift Naturwissenshaften :343-360. Gitelman SE, Kobrin MS, Ye JQ, Lopez AR, Lee A & Derynck R (1994) Recombinant Vgr-1/BMP6-expressing tumors induce fibrosis and endochondral bone formation in vivo. J Cell Biol 126:1595-1609. Goss RJ (1983a) A fawn's first antlers. In: Goss RJ (ed) Deer Antlers: Regeneration, Function, and Evolution. Academic Press, Inc., New York, p 122-136. Goss RJ (1983b) Developmental anatomy of antlers. In: Goss RJ (ed) Deer Antlers: Regeneration, Function, and Evolution. Academic Press, Inc., New York, p 137-171. Grant MH, Nugent C, & Bertrand R (1994) Studies on nickel-induced inhibition of fibroblast growth. Toxicol In Vitro 8:191-195.

58
Gray C, Boyde A & Jones SJ (1996) Topographically induced bone formation in vitro: implications for bone implants and bone grafts. Bone 18:115-123. Gray C, Hukkanen M, Konttinen YT, Terenghi G et al. (1992) Rapid neural growth: calcitonin generelated peptide and substance P-containing nerves attain exceptional growth rates in regenerating deer antler. Neuroscience 50:953-963. Gray C, Taylor M, Horton MA, Loudon ASI & Arnett TR (1992) Studies on Cells Derived from Growing Deer Antler. In: Brown RD (ed) The Biology of the deer. Springer-Verlag, New York, p 511-519. Grey A, Mitnick MA, Masiukiewicz U, Sun BH, Rudikoff S et al. (1999) A role for interleukin-6 in parathyroid hormone-induced bone resorption in vivo. Endocrinology 140:4683-4690. Griffith DL, Keck PC, Sampath TK, Rueger DC & Carlson WD (1996) Three-dimensional structure of recombinant human osteogenic protein 1: structural paradigm for the transforming growth factor beta superfamily. Proc Natl Acad Sci USA 93:878-883. Grimsrud CD, Romano PR, D'Souza M, Puzas JE, Reynolds PR, Rosier RN & O'Keefe RJ (1999) BMP-6 is an autocrine stimulator of chondrocyte differentiation. J Bone Miner Res 14:475-482. Gumbiner BM (1993) Proteins associated with the cytoplasmic surface of adhesion molecules. Neuron 11:551-564. Hanawa T & Ota M (1991) Calcium phosphate naturally formed on titanium in electrolyte solution. Biomaterials 12:767-774. Hansson S & Norton M (1999) The relation between surface roughness and interfacial shear strength for bone-anchored implants. A mathematical model. J Biomech 32:829-836. Hazama M, Aono A, Ueno N & Fujisawa Y (1995) Efficient expression of a heterodimer of bone morphogenetic protein subunits using a baculovirus expression system. Biochem Biophys Res Commun 209:859-866. Hildebrand HF & Hornez JC (1998) Biological response and biocompatibility. In: Helsen JA & Breme HJ (eds) Metals as biomaterials. John Wiley & Sons Ltd, Chichester, p 265-290. Hino J, Takao M, Takeshita N, Konno Y, Nishizawa T, Matsuo H & Kangawa K (1996) cDNA cloning and genomic structure of human bone morphogenetic protein- 3B (BMP-3b). Biochem Biophys Res Commun 223:304-310. Hirschman A, Deutsch D, Hirschman M, Bab IA, Sela J & Muhlrad A (1983) Neutral peptidase activities in matrix vesicles from bovine fetal alveolar bone and dog osteosarcoma. Calcif Tissue Int 35:791-797. Hohling HJ, Barckhaus RH, Krefting ER, Quint P & Althoff J (1978) Quantitative electron microscopy of the early stages of cartilage mineralization. Metab Bone Dis Relat Res 1:109-114. Humbeeck J, Stalmans R & Besselink PA (1998) Shape memory alloys. In: Helsen, JA & Breme HJ (ed) Metals as biomaterials. John Wiley & Sons Ltd, Chichester, England, p 73-100 Hunter A, Archer CW, Walker PS & Blunn GW (1995) Attachment and proliferation of osteoblasts and fibroblasts on biomaterials for orthopaedic use. Biomaterials 16:287-295. Ihbe A, Baumann G, Heinzmann U & Atkinson MJ (1998) Loss of the differentiated phenotype precedes apoptosis of ROS 17/2.8 osteoblast-like cells. Calcif Tissue Int 63:208-213. Ishidou Y, Kitajima I, Obama H, Maruyama I, Murata F et al. (1995) Enhanced expression of type I receptors for bone morphogenetic proteins during bone formation. J Bone Miner Res 10:16511659. Jilka RL, Weinstein RS, Bellido T, Parfitt AM & Manolagas SC (1998) Osteoblast programmed cell death (apoptosis): Modulation by growth factors and cytokines. J Bone Miner Res 13:793-802. Jones SJ, Gray C, Sakamaki H, Arora M, Boyde A, Gourdie R & Green C (1993) The incidence and size of gap junctions between the bone cells in rat calvaria. Anat Embryol (Berlin) 187:343-352. Jortikka L, Marttinen A & Lindholm TS (1993) Partially purified reindeer (Rangifer tarandus) bone morphogenetic protein has a high bone-forming activity compared with some other artiodactyls. Clin Orthop Rel Res 297:33-37.

59
Kapanen A, Birr E & Vnnen K (2000) Rangifer tarandus tarandus bone morphogenetic protein 3b mRNA, partial coding sequence. Genbank accession number AF300813. Kasemo B & Lausmaa J (1991) The biomaterial-tissue interface and its analogues in surface science and technology. In: Davies JE (ed) The bone-biomaterial interface. University of Toronto press, Toronto, p 19-32. Kierdorf H & Kierdorf U (2000) Roe deer antlers as monitoring units for assessing temporal changes in environmental pollution by fluoride and lead in a German forest area over a 67-year period. Arch Environ Contam Toxicol 39:1-6. Kierdorf U & Kierdorf H (2001) Fluoride concentrations in antler bone of roe deer (Capreolus capreolus) indicate decreasing fluoride pollution in an industrialized area of western Germany. Environ Toxicol Chem 20:1507-1510. Kim H & Johnson JW (1999) Corrosion of stainless steel, nickel-titanium, coated nickel-titanium, and titanium orthodontic wires. Angle Orthod 69:39-44. Kirkpatrick CJ, Mohr W & Haferkamp O (1982) The effects of nickel ions on articular chondrocyte growth in monolayer culture. Res Exp Med (Berlin) 181:259-264. Klein CB, Conway K, Wang XW, Bhamra RK, Lin XH, Cohen MD, Annab L, Barrett JC & Costa M (1991a) Senescence of nickel-transformed cells by an X chromosome: possible epigenetic control. Science 251:796-799. Klein CB, Frenkel B, & Costa M (1991b) The role of oxidative processes in metal carcinogenesis. Chem Res Toxicol 4:592-604. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K et al. (1997) Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755-764. Kurihara N, Bertolini D, Suda T, Akiyama Y & Roodman GD (1990) IL-6 stimulates osteoclast-like multinucleated cell formation in long term human marrow cultures by inducing IL-1 release. J Immunol 144:4226-4230. Layor PA, Revell PA, Gray AB, Wright S, Railton GT & Freeman MA (1991) Sensitivity to titanium. A case of implant failure? J Bone Joint Surg 73:25-28. Levander G (1938) A study of bone regeneration. Surg Gynecol Obstet 67:705-748. Majeska RJ, Rodan SB, Rodan GA (1980) Parathyroid hormone-responsive clonal cell lines from rat osteosarcoma. Endocrinology 107:1494-1503. Marks SC & Hermey DC (1996) Structure and development of bone. In: Bilezikian JP, Raisz LG, & Rodan GA (eds) Principles in bone biology. Academic Press, San Diego, p 3-14. Marotti G, Ferretti M, Muglia MA, Palumbo C & Palazzini S (1992) A quantitative evaluation of osteoblast-osteocyte relationships on growing endosteal surface of rabbit tibiae. Bone 13:363-8. Martin RB (2000a) Toward a unifying theory of bone remodelling. Bone 26:1-6. Martin RB (2000b) Does osteocyte formation cause the nonlinear refilling of osteons? Bone 26:7178. Mason DJ, Hillam RA & Skerry TM (1996) Constitutive in vivo mRNA expression by osteocytes of beta-actin, osteocalcin, connexin-43, IGF-I, c-fos and c-jun, but not TNF-alpha nor tartrate-resistant acid phosphatase. J Bone Miner Res 11:350-357. McKay GC, MacNair R, MacDonald C, & Grant MH (1996) Interactions of orthopaedic metals with an immortalized rat osteoblast cell line. Biomaterials 17:1339-1344. McPherron AC & Lee SJ (1993) GDF-3 and GDF-9: two new members of the transforming growth factor-beta superfamily containing a novel pattern of cysteines. J Biol Chem 268:3444-3449. Misra M, Rodriquez RE, & Kasprzak KS (1990) Nickel induced lipid peroxidation in the rat: correlation with nickel effect on antioxidant defence systems. Toxicology 64:1-17. Morais S, Sousa JP, Fernandes MH & Carvalho GS (1998) In vitro biomineralization by osteoblastlike cells. I. Retardation of tissue mineralization by metal salts. Biomaterials 19:13-21.

60
Nakase T, Nomura S, Yoshikawa H, Hashimoto J et al. (1994) Transient and localized expression of bone morphogenetic protein 4 messenger RNA during fracture healing. J Bone Miner Res 9:651659. Neuhaus H, Rosen V & Thies RS (1999) Heart specific expression of mouse BMP-10, a novel member of the TGF-beta superfamily. Mech Dev 80:181-184. Nichols KG & Puleo DA (1997) Effect of metal ions on the formation and function of osteoclastic cells in vitro. J Biomed Mater Res 35:265-271. Noda M & Camilliere JJ (1989) In vivo stimulation of bone formation by transforming growth factorbeta. Endocrinology 124:2991-2994. Oller AR, Costa M & Oberdorster G (1997) Carcinogenicity assessment of selected nickel compounds. Toxicol Appl Pharmacol 143:152-166. Otto F, Thornell AP, Crompton T, Denzel A, et al. (1997) Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89:765771. zkaynak E, Rueger DC, Drier EA, Corbett C, Ridge RJ, Sampath TK & Oppermann H (1990) OP1 cDNA encodes an osteogenic protein in the TGF-beta family. EMBO 9:2085-2093. Palumbo C, Palazzini S & Marotti G (1990) Morphological study of intercellular junctions during osteocyte differentiation. Bone 11:401-406. Parfitt AM (1990) Bone-forming cells in clinical conditions. In: Hall BK (ed) Bone, vol 1: Osteoblast and Osteocyte. Telford Press and CRC Press, Boca Raton, p 351-429. Peters MA, Schroeter AL, van Hale HM & Broadbent JC (1984) Pacemaker contact sensitivity. Contact Dermatitis 11:214-222. Price JS, Oyajobi BO, Nalin AM, Frazer A, Russell RG & Sandell LJ (1996) Chondrogenesis in the regenerating antler tip in red deer: expression of collagen types I, IIA, IIB, and X demonstrated by in situ nucleic acid hybridization and immunocytochemistry. Develop Dyn 205:332-347. Price JS, Oyajobi BO, Oreffo ROC & Russell RGG (1994) Cells cultured from the growing tip of red deer antler express alkaline phosphatase and proliferation in response to insulin-like growth factor-I. J Endocrin 143:R9-R16. Quarles LD, Hartle JE, Siddhanti SR, Guo R & Hinson TK (1997) A distinct cation-sensing mechanism in MC3T3-E1 osteoblasts functionally related to the calcium receptor. J Bone Miner Res 12:393-402. Riley EH, Lane JM, Urist MR, Lyons KM & Lieberman JR (1996) Bone morphogenetic protein-2: biology and applications. Clin Orthop Rel Res 39-46. Ripamonti U, Van Den HB, Crooks J, Tucker MM et al. (2000) Long-term evaluation of bone formation by osteogenic protein 1 in the baboon and relative efficacy of bone-derived bone morphogenetic proteins delivered by irradiated xenogeneic collagenous matrices. J Bone Miner Res 15:1798-1809. Ripamonti U, Van Den HB, Sampath TK, Tucker MM, Rueger DC & Reddi AH (1996) Complete regeneration of bone in the baboon by recombinant human osteogenic protein-1 (hOP-1, bone morphogenetic protein-7). Growth Factors 13:273-289. Robey PG, Young MF, Flanders KC, Roche NS, Kondaiah P et al. (1987) Osteoblasts synthesize and respond to transforming growth factor-type beta (TGF-beta) in vitro. J Cell Biol 105:457-463. Rodriquez RE, Misra M, & Kasprzak KS (1990) Effects of nickel on catalase activity in vitro and in vivo. Toxicology 63:45-52. Rolf HJ & Enderle A (1999) Hard fallow deer antler: a living bone till antler casting? Anatomic Rec 255:69-77. Rondelli G & Vicentini B (1999) Localized corrosion behaviour in simulated human body fluids of commercial Ni-Ti orthodontic wires. Biomaterials 20:785-792.

61
Roodman GD (1992) Interleukin-6: an osteotropic factor? J Bone Miner Res 7:475-478. Rubinacci A, Villa I, Dondi BF, Borgo E, Ferretti M, Palumbo C & Marotti G (1998) Osteocyte-bone lining cell system at the origin of steady ionic current in damaged amphibian bone. Calcif Tissue Int 63:331-339. Ryhnen J, Kallioinen M, Serlo W, Permki P, Junila J et al. (1999a) Bone healing and mineralization, implant corrosion and trace metals after nickel-titanium shape memory metal intramedullary fixation. J Biomed Mat Res 47:472-480. Ryhnen J, Kallioinen M, Tuukkanen J, Junila J, Niemel E, Sandvik P & Serlo W (1998) In vivo biocompatibility evaluation of nickel-titanium shape memory metal alloy: muscle and perineural tissue responses and encapsule membrane thickness. J Biomed Mat Res 41:481-488. Ryhnen J, Kallioinen M, Tuukkanen J, Lehenkari P, Junila J et al. (1999b) Bone modeling and cellmaterial interface responses induced by nickel-titanium shape memory alloy after periosteal implantation. Biomaterials 20:1309-1317. Ryhnen J, Niemi E, Serlo W, Niemel E, Sandvik P, Pernu H & Salo T (1997) Biocompatibility of nickel-titanium shape memory metal and its corrosion behavior in human cell cultures. J Biomed Mat Res 35:451-457. Sampath TK, Coughlin JE, Whetstone RM, Banach D, Corbett C et al. (1990) Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-beta superfamily. J Biol Chem 265:13198-13205. Sarkar NK, Redmond W, Schwaninger B & Goldberg AJ (1983) The chloride corrosion behaviour of four orthodontic wires. J Oral Rehabil 10:121-128. Schedle A, Samorapoompichit P, Rausch-Fan XH, Franz A et al. (1995) Response of L-929 fibroblasts, human gingival fibroblasts, and human tissue mast cells to various metal cations. J Dent Res 74:1513-1520. Serre CM, Boivin G, Obrant KJ & Linder L (1994) Osseointegration of titanium implants in the tibia. Electron microscopy of biopsies from 4 patients. Acta Orthop Scand 65:323-327. Shabalovskaya SA (1996) On the nature of the biocompatibility and on medical applications of NiTi shape memory and superelastic alloys. Biomed Mat Eng 6:267-289. Shabalovskaya SA & Anderegg JW (1995) Surface spectroscopic characterization of NiTi nearly equiatomic shape memory alloys for implants. J Vac Sci Technol A13:2624-2632. Shida J, Trindade MC, Goodman SB, Schurman DJ & Smith RL (2000) Induction of interleukin-6 release in human osteoblast-like cells exposed to titanium particles in vitro. Calcif Tissue Int 67:151-155. Simon M, Maresh JG, Harris SE, Hernandez JD, Arar M, Olson MS & Abboud HE (1999) Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney. Am J Physiol 276:F382-F389. Simske SJ & Sachdeva R (1995) Cranial bone apposition and ingrowth in a porous nickel-titanium implant. J Biomed Mat Res 29:527-533. Skalak R & Zhao Y (2000) Similarity of stress distribution in bone for various implant surface roughness heights of similar form. Clin Implant Dent Relat Res 2:225-230. Song JJ, Celeste AJ, Kong FM, Jirtle RL, Rosen V & Thies RS (1995) Bone morphogenetic protein9 binds to liver cells and stimulates proliferation. Endocrinology 136:4293-4297. Sousa SR & Barbosa MA (1996) Effect of hydroxyapatite thickness on metal ion release from Ti6Al4V substrates. Biomaterials 17:397-404. Speck KM & Fraker AC (1980) Anodic polarization behavior of Ti-Ni and Ti-6A1-4V in simulated physiological solutions. J Dent Res 59:1590-1595. Stanford CM, Keller JC & Solursh M (1994) Bone cell expression on titanium surfaces is altered by sterilization treatments. J Dent Res 73:1061-1071.

62
Stein GS, Lian J, Stein JL, vanWijnen AJ, Frenkel B & Montecino M (1996) Mechanisms regulating osteoblast proliferation and differentiation. In: Bilezikian JP, Raisz LG, & Rodan GA (eds) Principles of bone biology. Academic Press, San Diego, p 69-86. Storm EE, Huynh TV, Copeland NG, Jenkins NA, Kingsley DM & Lee SJ (1994) Limb alterations in brachypodism mice due to mutations in a new member of the TGF-beta superfamily. Nature 368:639-642. Sunderman FW, Jr., Hopfer SM, Knight JA, McCully KS et al. (1987) Physicochemical characteristics and biological effects of nickel oxides. Carcinogenesis 8:305-313. Suttie JM & Fennessy PF (1990) Antler regeneration - studies with antler removal, axial topography and angiography. In: Bubenik A & Bubenik G (eds) Antlers, Horns, Cranial Appendages of Pecoran. Springer-Verlag, New York, p 313-338. Suttie JM, Fennessy PF, Mackintosh CG, Corson ID, Christie R & Heap SW (1985) Sequential cranial angiography of young red deer stags. In Fennessy PF; Drew K. (eds) Biology of Deer Production. Research Society of New Zealand, Wellington, p 263-268. Takao M, Hino J, Takeshita N, Konno Y, Nishizawa T, Matsuo H & Kangawa K (1996) Identification of rat bone morphogenetic protein-3b (BMP-3b), a new member of BMP-3. Biochem Biophys Res Commun 219:656-662. Takeshita F, Takata H, Ayukawa Y & Suetsugu T (1997) Histomorphometric analysis of the response of rat tibiae to shape memory alloy (nitinol). Biomaterials 18:21-25. Thiede MA, Yoon K, Golub EE, Noda M & Rodan GA (1988) Structure and expression of rat osteosarcoma (ROS 17/2.8) alkaline phosphatase: product of a single copy gene. Proc Natl Acad Sci U S A 85:319-323. Thompson GJ & Puleo DA (1995) Effects of sublethal metal ion concentrations on osteogenic cells derived from bone marrow stromal cells. J Appl Biomater 6:249-258. Thompson GJ & Puleo DA (1996) Ti-6Al-4V ion solution inhibition of osteogenic cell phenotype as a function of differentiation timecourse in vitro. Biomaterials 17:1949-1954. Trentz OA, Zellweger R, Amgwerd MG & Uhlschmid GK (1997) Testing bone implants in cell lines and human osteoblasts. Unfallchirurg 100:39-43. Urist M (1965) Bone: Formation by autoinduction. Science 150:893-899. Vehof JW, Spauwen PH & Jansen JA (2000) Bone formation in calcium-phosphate-coated titanium mesh. Biomaterials 21:2003-2009. Viljanen VV, Gao TJ, Marttinen A & Lindholm TS (1996) Partial purification and characterization of bone morphogenetic protein from bone matrix of the premature moose (Alces alces): degradation of bone-inducing activity during storage. Eur Surg Res 28:447-460. Vnnen K (1996) Osteoclast function: Biology and mechanisms. In: Bilezikian JP, Raisz LG, & Rodan GA (eds) Principles of bone biology. Academic press, San Diego, p 103-114. Wang EA, Rosen V, D'Alessandro JS, Bauduy M, Cordes P et al. (1990) Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci U S A 87:2220-2224. Wataha IC, Lockwood PE, Marek M & Ghazi M (1999) Ability of Ni-containing biomedical alloys to activate monocytes and endothelial cells in vitro. J Biomed Mat Res 45:251-257. Wataha JC, Craig RG & Hanks CT (1991) The release of elements of dental casting alloys into cellculture medium. J Dent Res 70:1014-1018. Wataha JC, Hanks CT & Craig RG (1993) The effect of cell monolayer density on the cytotoxicity of metal ions which are released from dental alloys. Dent Mater 9:172-176. Wetterwald A, Hoffstetter W, Cecchini MG, Lanske B, Wagner C, Fleisch H & Atkinson M (1996) Characterization and cloning of the E11 antigen, a marker expressed by rat osteoblasts and osteocytes. Bone 18:125-132. Wever DJ, Veldhuizen AG, de Vries J, Busscher HJ, Uges DR & van Horn JR. (1998) Electrochemical and surface characterization of a nickel- titanium alloy. Biomaterials 19:761-769.

63
Wever DJ, Veldhuizen AG, Sanders MM, Schakenraad JM & van Horn JR (1997) Cytotoxic, allergic and genotoxic activity of a nickel-titanium alloy. Biomaterials 18:1115-1120. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM & Wang EA (1988) Novel regulators of bone formation: molecular clones and activities. Science 242:15281534. Wuthier RE, Chin JE, Hale JE, Register TC, Hale LV & Ishikawa Y (1985) Isolation and characterization of calcium-accumulating matrix vesicles from chondrocytes of chicken epiphyseal growth plate cartilage in primary culture. J Biol Chem 260:15972-15979. Yamamoto A, Honma R & Sumita M (1998) Cytotoxicity evaluation of 43 metal salts using murine fibroblasts and osteoblastic cells. J Biomed Mater Res 39:331-340. Yokoyama K, Hamada K, Moriyama K & Asaoka K (2001) Degradation and fracture of Ni-Ti superelastic wire in an oral cavity. Biomaterials 22:2257-2262. Zhao GQ, Deng K, Labosky PA, Liaw L & Hogan BL (1996) The gene encoding bone morphogenetic protein 8B is required for the initiation and maintenance of spermatogenesis in the mouse. Genes Dev 10:1657-1669. Zhao R, Lawler AM & Lee SJ (1999) Characterization of GDF-10 expression patterns and null mice. Dev Biol 212:68-79. Zitter H & Plenk H, Jr. (1987) The electrochemical behavior of metallic implant materials as an indicator of their biocompatibility. J Biomed Mat Res 21:881-896.