ACTA EFFECT OF ESTROGEN AND DIETARY LOADING ON RAT CONDYLAR CARTILAGE UNIVERSITATIS OULUENSIS D Marko Orajärvi MEDICA OULU PDF

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OULU 2015 D 1283 ACTA Marko Orajärvi UNIVERSITATIS OULUENSIS D MEDICA EFFECT OF ESTROGEN AND DIETARY LOADING ON RAT CONDYLAR CARTILAGE UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF

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OULU 2015 D 1283 ACTA Marko Orajärvi UNIVERSITATIS OULUENSIS D MEDICA EFFECT OF ESTROGEN AND DIETARY LOADING ON RAT CONDYLAR CARTILAGE UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF MEDICINE, INSTITUTE OF DENTISTRY, DEPARTMENT OF ORAL DEVELOPMENT AND ORTHODONTICS, DEPARTMENT OF PROSTHETIC DENTISTRY AND STOMATOGNATHIC PHYSIOLOGY; FINNISH DOCTORAL PROGRAM IN ORAL SCIENCES FINDOS; MEDICAL RESEARCH CENTER OULU; OULU UNIVERSITY HOSPITAL ACTA UNIVERSITATIS OULUENSIS D Medica 1283 MARKO ORAJÄRVI EFFECT OF ESTROGEN AND DIETARY LOADING ON RAT CONDYLAR CARTILAGE Academic dissertation to be presented with the assent of the Doctoral Training Committee of Health and Biosciences of the University of Oulu for public defence in the Leena Palotie auditorium (101A) of the Faculty of Medicine (Aapistie 5 A), on 27 February 2015, at 12 noon UNIVERSITY OF OULU, OULU 2015 Copyright 2015 Acta Univ. Oul. D 1283, 2015 Supervised by Professor Pertti Pirttiniemi Professor Aune Raustia Reviewed by Professor Timo Peltomäki Professor Mauno Könönen Opponent Professor Stavros Kiliaridis ISBN (Paperback) ISBN (PDF) ISSN (Printed) ISSN (Online) Cover Design Raimo Ahonen JUVENES PRINT TAMPERE 2015 Orajärvi, Marko, Effect of estrogen and dietary loading on rat condylar cartilage. University of Oulu Graduate School; University of Oulu, Faculty of Medicine, Institute of Dentistry, Department of Oral Development and Orthodontics, Department of Prosthetic Dentistry and Stomatognathic Physiology; Finnish Doctoral Program in Oral Sciences FINDOS; Medical Research Center Oulu; Oulu University Hospital Acta Univ. Oul. D 1283, 2015 University of Oulu, P.O. Box 8000, FI University of Oulu, Finland Abstract The temporomandibular joint (TMJ) is a synovial joint which attaches the mandible to the skull. The head of the mandibular condyle is covered by condylar cartilage, which functions as both growth and articular cartilage. Masticatory forces are transmitted to the condylar cartilage, and the consistency of a person s diet partly defines the loading force. Condylar cartilage acts as a loadabsorbing structure together with the articular disc. Temporomandibular disorders (TMDs) are a wide group of pathological conditions involving pain and dysfunction in the masticatory system. Females comprise the majority of patients, having more symptoms and clinical signs than men. Because of this gender distribution, it has been suggested that the female sex hormone estrogen has an influence on the pathogenesis of TMJ osteoarthrosis. However, little is known about how estrogen affects condylar cartilage. The effect of estrogen deficiency and altered dietary loading on condylar cartilage was studied with female rats, which were distributed to four groups depending on ovariectomy and diet consistency, and further to two distinct age groups. Expression of type II and X collagens, adiponectin, ERα, MMP-3, MMP-8, TRAP, and cathepsin K was investigated. Markers of apoptosis and proliferation of cartilage cells were investigated. A lack of estrogen increased and a soft diet decreased the thickness of condylar cartilage. Expression of proliferating cell nuclear antigen (PCNA, proliferation marker) was higher in ovariectomized rats fed a normal diet when compared with control rats. The area of type II and X collagens in condylar cartilage was larger when estrogen was not present and smaller when the diet consistency was soft. Expression of MMP-3 was higher in ovariectomized rats than in control rats. Ovariectomized rats fed a soft diet had higher expression of MMP-8 than ovariectomized rats fed a normal diet. However, control rats fed a soft diet had lower proportional expression of MMP- 8 than normal diet controls. The proportional amount of cartilage cells stained against adiponectin was higher in rats fed a soft diet when compared with rats fed a normal diet. The proportional amount of cartilage cells expressing ERα was higher in ovariectomized rats than in control rats. Osteoclast markers cathepsin K and TRAP showed that ovariectomized rats had fewer osteoclasts than control rats had. The average size of osteoclasts was smaller in ovariectomized rats when compared with control rats. The results of this study show that estrogen and altered dietary loading have an effect on condylar cartilage. Further studies are needed to evaluate the significance of these changes, and especially their association with TMJ disorders. Keywords: condylar cartilage, condyle, estrogen, loading, temporomandibular disorders, tmj Orajärvi, Marko, Estrogeenin ja purentarasituksen vaikutus rotan leukanivelrustoon. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta, Hammaslääketieteen laitos, Hampaiston kehitys- ja oikomisoppi, Hammasproteesioppi ja kliininen purentafysiologia; Suun terveystieteiden tohtoriohjelma FINDOS; Medical Research Center Oulu; Oulun yliopistollinen sairaala Acta Univ. Oul. D 1283, 2015 Oulun yliopisto, PL 8000, Oulun yliopisto Tiivistelmä Leukanivel on synoviaalinivel, joka liittää mandibulan eli alaleuan kalloon. Alaleuan nivellisäkkeen eli kondyylin päässä oleva rustokerros toimii sekä kasvu- että nivelrustona. Purentavoimat välittyvät kondyylirustoon, ja kuormituksen määrä on riippuvainen ruoan kovuusasteesta. Kondyylirusto toimii kuormituksen vastaanottajana yhdessä nivelvälilevyn kanssa. Purentaelimistön toimintahäiriöihin (TMD) kuuluu laaja joukko patologisia tiloja, joihin liittyy kipua ja dysfunktiota purentaelimistössä. Potilaista suurin osa on naisia, ja heillä on oireita sekä sairauden kliinisiä tunnusmerkkejä enemmän kuin miehillä. Sukupuolijakaumasta johtuen on arveltu, että naissukupuolihormoni estrogeenilla voisi olla vaikutusta leukanivelartroosin patogeneesiin. Tällä hetkellä tietoa on hyvin vähän siitä, miten estrogeeni vaikuttaa leukanivelrustoon. Estrogeenin puutoksen ja muunnellun purentarasituksen vaikutusta leukanivelrustoon tutkittiin naarasrotilla, jotka jaettiin neljään eri ryhmään munasarjojen poiston ja dieetin kovuusasteen mukaan sekä lisäksi kahteen eri ikäryhmään. Tyypin II ja X kollageenin, adiponektiinin, estrogeenireseptori α:n, MMP-3:n, MMP-8:n, TRAP:n ja katepsiini K:n ekspressio tutkittiin. Rustosoluista tutkittiin apoptoosin ja proliferaation merkkiaineet. Estrogeenin puute lisäsi ja pehmeä dieetti pienensi kondyyliruston paksuutta. PCNA:n (proliferating cell nuclear antigen) ekspressio oli suurempi normaalilla dieetillä olleilla ovariapoistetuilla rotilla kuin kontrollirotilla. Tyypin II ja X kollageenien osuus oli suurempi ovariapoistettujen rottien kondyylirustossa kuin pehmeällä dieetillä olleilla. MMP-3:n ekspressio oli korkeampi ovariapoistetuilla rotilla kuin kontrolleilla. Ovariapoistetuilla rotilla, jotka olivat pehmeällä dieetillä, oli suurempi MMP-8:n ekspressio kuin normaalilla dieetillä olleilla ovariapoistetuilla rotilla. Kuitenkin MMP-8:n ekspressio oli pienempi pehmeällä dieetillä olleilla kontrollirotilla kuin normaalilla dieetillä olleilla. Adiponektiinin tunnistavalle vasta-aineelle positiivisten solujen suhteellinen lukumäärä oli suurempi pehmeällä dieetillä olleilla rotilla kuin normaalilla dieetillä olleilla. Estrogeenireseptori α:a ekspressoivien solujen suhteellinen lukumäärä oli suurempi ovariapoistetuilla rotilla kuin kontrolleilla. Osteoklastien merkkiaineet katepsiini K ja TRAP osoittivat, että ovariapoistetuilla rotilla oli vähemmän osteoklasteja kuin kontrollirotilla. Osteoklastien keskimääräinen koko oli pienempi ovariapoistetuilla rotilla kuin kontrolleilla. Tutkimuksen tulokset osoittavat, että estrogeeni ja muunneltu purentarasitus vaikuttavat leukanivelrustoon. Lisätutkimuksia tarvitaan, jotta voidaan arvioida näiden muutosten merkitsevyys ja erityisesti niiden yhteys leukanivelen toimintahäiriöihin. Asiasanat: estrogeeni, kondyyli, kondyylirusto, leukanivel, purentaelimistön toimintahäiriöt, purentarasitus Acknowledgements This research was carried out at the Department of Prosthetic Dentistry and Stomatognathic Physiology and Department of Oral Development and Orthodontics, Institute of Dentistry, Faculty of Medicine, University of Oulu. Part of this work was carried out at the Institute of Biotechnology, University of Helsinki. This research was carried out in co-operation with the Finnish Doctoral Program in Oral Sciences (FINDOS). I am sincerely grateful to my supervisors, Professor Aune Raustia D.D.S, Ph.D, and Professor Pertti Pirttiniemi, D.D.S, Ph.D. They introduced me to research and have taught me both practical and scientific matters throughout the work. Aune Raustia has given me great insight into temporomandibular physiology and pathology and has always made time to focus on my often most basic questions. Pertti Pirttiniemi has offered his expertise in the development and the growth of condyle and has been both encouraging and patient with my work. I learned a great deal during my relatively short time spent at the Laboratory Animal Centre in Oulu. I wish to thank all the personnel working there. I was privileged to work with Sakari Laaksonen D.V.M, who has since then taught me a lot about research in general. The largest part of the time gone into this research was spent in our histological laboratory at the Institute of Dentistry with Ms Maritta Harjapää, Ms Tanja Kuusisto, Ms Sanna Juntunen, Ms Merja Tyynismaa, and Ms Eeva-Maija Kiljander. Warmest thanks belong to them for guiding me in the laboratory and for being my friends. I wish to thank Professor Tuula Salo, D.D.S, Ph.D, and Professor Leo Tjäderhane D.D.S, Ph.D, for the inspirational discussions on science. I wish to thank all the personnel in the research group of Irma Thesleff, D.D.S, Ph.D, at the Institute of Biotechnology, University of Helsinki for their input. I wish to express my gratitude to the referees of this thesis, Professor Timo Peltomäki and Professor Mauno Könönen, for their honest and meticulous review of the manuscript. I am indebted to the co-authors of the articles: Elina Puijola, D.D.S, Outi Hirvonen, D.D.S, Petri Tiilikainen, D.D.S, Ms Henna Hartikainen, Xiaodong Liu, D.D.S, Ph.D, Shi-Bin Yu, M.D.S, Ph.D, Professor Meiqing Wang, D.D.S, M.D, Ph.D, and Professor Irma Thesleff, D.D.S, Ph.D. I would like to thank my colleagues Egle Jonaviciute, D.D.S, Ms Riikka Hauru and Ms Heidi Mäki-Kojola, who helped greatly in the preparation of samples. 7 I want to acknowledge the excellent work and experience of the statisticians, Ahti Niinimaa, Ph.D, and Paula Pesonen, M.Sc. Mr Reijo Kettunen is acknowledged for his technical assistance and the schematic drawing of the condyle in one article. The thesis and three articles would not have been published without the revisions made by Mr Keith Kosola, whom I wish to thank very much. I thank the people in my follow-up group: Professor Tellervo Tervonen, D.D.S, Ph.D, and Docent Tuomo Heikkinen, D.D.S, Ph.D. Thanks are also due to my colleague Anniina Haro, D.D.S, for the numerous discussions and encouragement during the research. Her bringing me food when I was ill is acknowledged. Mr Juhani Vänttilä, Hans Tuomas, D.D.S, Timo Ruotsalainen, D.D.S, and Mr Eino Kemppainen have helped me out in many ways at the University of Oulu. Thanks are due to them for their inspiration and for making the everyday life at the University more enjoyable. This study has been financially supported by the FINDOS, Oulu University Hospital funding, and research grants from the Finnish Dental Society Apollonia. The grants are acknowledged. I hold my parents and grandmother Tellervo in the highest regard for supporting me throughout these 29 years. I thank my brother Mika and his wife Outi, who have supported me in many ways during my studies. My wife s family also deserves a great deal of appreciation from me for helping me out. Finally, I express my heartfelt gratitude and love to my adorable wife Helena, who made this book possible. 15 th January, 2014 Marko Orajärvi 8 Abbreviations ANOVA Analysis of variance AP-1 Activator protein 1 BMD Bone mineral density BSA Bovine serum albumin CDMP1 Cartilage-derived morphogenic protein 1 ECM Extracellular matrix ER Estrogen receptor FSH Follicle-stimulating hormone HSD Honestly significant difference ICC Intraclass correlation coefficient IL Interleukin KO Knockout LH Luteinizing hormone M-CSF Macrophage colony-stimulating factor MMP Matrix metalloproteinase mrna Messenger ribonucleic acid OA Osteoarthrosis OPG Osteoprotegerin PBS Phosphate-buffered saline PCNA Proliferating cell nuclear antigen PTHrP Parathyroid hormone-related protein RANK Receptor activator of nuclear factor kappa-b RANKL Receptor activator of nuclear factor kappa-b ligand RNA Ribonucleic acid RUNX Runt-related transcription factor 2 TIMP Tissue inhibitor of metalloproteinase TMJ Temporomandibular joint TMD Temporomandibular disorders TNF Tumor necrosis factor TRAP Tartrate-resistant acid phosphatase TUNEL Terminal deoxynucleotidyl transferase dutp nick end labeling UTP Uridine 5'-triphosphate VEGF Vascular endothelial growth factor 9 10 List of original articles This thesis is based on the following publications, which are referred throughout the text by their Roman numerals: I Orajärvi M, Puijola E, Yu SB, Liu X, Tiilikainen P, Wang M, Raustia A, Pirttiniemi P (2012) Effect of estrogen and dietary loading on condylar cartilage. J Orofac Pain 26: II Orajärvi M, Hirvonen O, Yu SB, Liu X, Tiilikainen P, Wang M, Raustia A, Pirttiniemi P (2011) Effect of estrogen and altered diet hardness on the expression of estrogen receptor alpha and matrix metalloproteinase-8 in rat condylar cartilage. J Orofac Pain 25: III Orajärvi M, Thesleff I, Hartikainen H, Raustia A, Pirttiniemi P. Effect of estrogen and food hardness on metabolism and turnover of condylar cartilage. J Oral Facial Pain Headache (In press). 11 12 Contents Abstract Tiivistelmä Acknowledgements 7 Abbreviations 9 List of original articles 11 Contents 13 1 Introduction 15 2 Review of literature Anatomy and function of the temporomandibular joint (TMJ) Temporomandibular joint of the rat Mandibular condylar cartilage Mechanical loading of the temporomandibular joint Collagens of condylar cartilage Temporomandibular disorders (TMDs) Role of gender in temporomandibular disorders Estrogen Estrogen in bone remodeling Estrogen in cartilage remodeling Matrix metalloproteinases (MMPs) and adiponectin Aims of the study 33 4 Materials and methods Experimental animals Ovariectomy Preparation of tissues Histological procedures Immunohistological procedures In situ hybridization Enzyme histochemistry (TRAP) TUNEL labeling Analysis Histomorphometric analysis Analysis of immunohistochemistry, in situ hybridization, and TRAP staining Statistical analysis Results 43 13 5.1 Changes in the histomorphology of condylar cartilage Immunohistochemistry Type II collagen Type X collagen Estrogen receptor alpha (ERα) TRAP and cathepsin K MMP-3 and MMP PCNA Adiponectin Apoptosis In situ hybridization with a type X collagen probe Discussion Role of estrogen and altered dietary loading in condylar cartilage remodeling Estrogen in bone metabolism Estrogen in cartilage remodeling Differences between condylar cartilage and other cartilages of different joints Estrogen receptors of condylar cartilage Collagens of condylar cartilage Matrix metalloproteinases in cartilage remodeling Proliferative activity in condylar cartilage Adiponectin Apoptosis of chondrocytes Adaptive capacity of condylar cartilage Summary and conclusions 67 References 69 Original articles 91 14 1 Introduction The temporomandibular joint (TMJ) is a synovial joint enclosed by a fibrous capsule, and it attaches the mandible to the skull. The head of the mandibular condyle is covered by condylar cartilage, which functions as both growth and articular cartilage. Temporomandibular disorders (TMDs) are a wide group of pathological conditions involving pain and dysfunction in the masticatory system. The etiology of TMD is considered multifactorial, including female gender, traumas involving the head and mandible, and psychological factors. Studies have shown that females comprise the majority of TMD patients, having more symptoms and clinical signs than men. Because of this gender distribution, it has been suggested that the female sex hormone influences the pathogenesis of TMDs. Masticatory forces are transmitted to the condylar cartilage, and the hardness of a person s diet partly defines the loading force. Condylar cartilage and the articular disc act as load-absorbing structures. Condylar cartilage is sensitive to loading, and an optimal level of loading is essential for condylar cartilage growth, maintaining both ideal proliferation and matrix production of chondrocytes. It can be suggested that sufficiently thick condylar cartilage is advantageous in maintaining the functional structure, because it has more strength and resiliency to withstand the forces created by mastication. Changes in the structure of mandibular condylar cartilage could compromise the normal function of the TMJ by altering the capability of condylar cartilage to tolerate mechanical forces, and thereby leading to degenerative changes. In this work the effect of estrogen deficiency and altered dietary loading on condylar cartilage was studied experimentally. The experimental animals were distributed to four groups depending on ovariectomy and diet consistency, and further to two distinct age groups. Expression of type II and X collagens, PCNA, adiponectin, ERα, MMP-3, MMP-8, TRAP, and cathepsin K was studied in order to investigate the mechanisms responsible for the changes in condylar cartilage caused by estrogen deficiency and altered dietary loading. 15 16 2 Review of literature 2.1 Anatomy and function of the temporomandibular joint (TMJ) The temporomandibular joint (TMJ) is a synovial joint enclosed by a fibrous capsule, and it is the joint that attaches the mandible to the skull (Enlow 1990, Okeson 2008). The mandibular condyle articulates at the base of the skull in a concave portion of the temporal bone the glenoid fossa (Fig. 1) (Okeson 2008). The articular disc separates these two bones forming superior and inferior joint cavities and functions as a third component of the joint, allowing the TMJ to perform various movements. The inner linings of the joint cavities are composed of specialized endothelial cells (synovial lining) that produce synovial fluid along with the synovial fringe located posteriorly to the articular disc. Fig. 1. A schematic illustration of the human TMJ. A) condyle, B) condylar cartilage, C) articular disc, D) glenoid fossa, E) superior synovial cavity, F) inferior synovial cavity, G) superior lateral pterygoid muscle, H) inferior lateral pterygoid muscle, I) retrodiscal tissues. 17 The articular surfaces of the condyle and glenoid fossa are lined with dense fibrous connective tissue, unlike other joints in the human body where hyaline cartilage constitutes a major part of the articular surface. The TMJ is classified as a ginglymoarthrodial joint, for it provides hinging movement in the sagittal plane, and also gliding movement, which is why the TMJ is regarded as both a ginglymoid and an arthrodial joint (Okeson 2008). The TMJ is important for chewing, swallowing and talking, and it is probably one of the most complex and frequently loaded joints in the human body. The movements of the mandible in TMJ function are restricted by bony articular surfaces together with masticatory muscles (Brown 1975). Further, the articular surfaces are separated by the articular disc, which moves more or less freely between the condyle and glenoid fossa and interacts with TMJ function a great deal (Isberg & Westesson 1998). 2.2 Temporomandibular joint of the rat The rat TMJ shows great similarity to the human TMJ both macroscopically and histologically (Fig. 2) (Porto
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