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DISSERTATIONS | MARI HUTTU | CELL – TISSUE INTERACTIONS IN NORMAL, AGING AND... | No 280
MARI HUTTU
CELL – TISSUE INTERACTIONS IN NORMAL, AGING AND OSTEOARTHRITIC ARTICULAR CARTILAGE
PUBLICATIONS OFTHE UNIVERSITY OF EASTERN FINLAND
Some symptoms associated with osteoarthritis are similar to changes in normal aging. However,
knowledge of the pathology of osteoarthritis at the cell and tissue level and how aging affects chondrocyte responses are still unclear. This thesis offers new information how aging affects chondrocyte
deformation processes at different depth of cartilage, and how they are controlled by tissue structure.
Further, the results of this thesis deepen the knowledge of changes in cell and tissue properties in cartilage during the progression of osteoarthritis.
This information could be used in planning of rehabilitation or treatment strategies for patients.
MARI HUTTU
CELL – TISSUE INTERACTIONS IN NORMAL, AGING AND
OSTEOARTHRITIC ARTICULAR
CARTILAGE
Mari Huttu
CELL – TISSUE INTERACTIONS IN NORMAL, AGING AND
OSTEOARTHRITIC ARTICULAR CARTILAGE
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences
No 280
University of Eastern Finland Kuopio
2017
Academic dissertation
To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium TTA in Tietoteknia Building at the University of Eastern Finland, Kuopio, on September, 22, 2017, at
12 o’clock noon
Mari Huttu
CELL – TISSUE INTERACTIONS IN NORMAL, AGING AND
OSTEOARTHRITIC ARTICULAR CARTILAGE
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences
No 280
University of Eastern Finland Kuopio
2017
Academic dissertation
To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium TTA in Tietoteknia Building at the University of Eastern Finland, Kuopio, on September, 22, 2017, at
12 o’clock noon
Grano Oy Jyväskylä, 2017
Editors: Pertti Pasanen, Matti Vornanen, Jukka Tuomela, Matti Tedre
Distribution: University of Eastern Finland / Sales of publications www.uef.fi/kirjasto
ISBN: 978-952-61-2589-3 (nid.) ISBN: 978-952-61-2590-9 (PDF)
ISSNL: 1798-5668 ISSN: 1798-5668 ISSN: 1798-5676 (PDF)
Author’s address: Mari Huttu
University of Eastern Finland Department of Applied Physics P.O.Box 1627
70211 KUOPIO FINLAND
email: Mari.Huttu@uef.fi Supervisors: Professor Rami Korhonen
University of Eastern Finland Department of Applied Physics P.O. Box 1627
70211 KUOPIO, FINLAND email: Rami.Korhonen@uef.fi Professor Mikko Lammi Umeå University
Department of Integrative Medical Biology SE-90187 UMEÅ, SWEDEN
email: mikko.lammi@umu.se Reviewers: Holly Leddy, Ph.D.
Duke University
Shared Materials Instrumentation Facility Box 90271
Durham, NC 27708
email: holly.leddy@duke.edu
Associate professor Susanna Miettinen University of Tampere
Faculty of Medicine and Life Sciences 33014 TAMPERE, FINLAND
email: susanna.miettinen@uta.fi Opponent: Professor Juha Tuukkanen
University of Oulu Medical Research Center P.O. Box 8000
90014 OULU, FINLAND email: juha.tuukkanen@oulu.fi
Author’s address: Mari Huttu
University of Eastern Finland Department of Applied Physics P.O.Box 1627
70211 KUOPIO FINLAND
email: Mari.Huttu@uef.fi Supervisors: Professor Rami Korhonen
University of Eastern Finland Department of Applied Physics P.O. Box 1627
70211 KUOPIO, FINLAND email: Rami.Korhonen@uef.fi Professor Mikko Lammi Umeå University
Department of Integrative Medical Biology SE-90187 UMEÅ, SWEDEN
email: mikko.lammi@umu.se Reviewers: Holly Leddy, Ph.D.
Duke University
Shared Materials Instrumentation Facility Box 90271
Durham, NC 27708
email: holly.leddy@duke.edu
Associate professor Susanna Miettinen University of Tampere
Faculty of Medicine and Life Sciences 33014 TAMPERE, FINLAND
email: susanna.miettinen@uta.fi Opponent: Professor Juha Tuukkanen
University of Oulu Medical Research Center P.O. Box 8000
90014 OULU, FINLAND email: juha.tuukkanen@oulu.fi
Huttu, Mari
Cell – tissue interactions in normal, aging and osteoarthritic articular cartilage Kuopio: University of Eastern Finland, 2017
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences 2017; 280 ISBN: 978-952-61-2589-3 (print)
ISSNL: 1798-5668 ISSN: 1798-5668
ISBN: 978-952-61-2590-9 (PDF) ISSN: 1798-5676 (PDF)
ABSTRACT
Articular cartilage is white, smooth, almost frictionless tissue without nerves or blood vessels covering ends of the long bones. It consists of one type of cells, chondrocytes, and extracellular matrix (ECM) comprising mainly tissue fluid, collagens and proteoglycans (PGs). The role of chondrocytes is to maintain the ECM.
Osteoarthritis (OA) is the most common joint disease in the world causing joint pain and decreased mobility of the joint. Age, gender, genetics, nutrition, obesity and abnormal joint loading are examples of risk factors of OA. Many early changes or symptoms, such as reduction of PG and disorganization of collagen fibrils, associated with OA are similar to changes in normal aging. On the other hand, aging also affects chondrocyte metabolism, and results in advanced glycation end products (AGEs) congregating the ECM. AGEs increase collagen cross-linking and ECM stiffness. Knowledge of the pathology OA and its progress is fairly well understood. However, at the cell and tissue level, many features are still unclear.
For example, which structural components and biomechanical properties of articular cartilage correlate together and participate in regulation of cell shape and volume in OA, are still unknown. It is also not known, how aging or induced collagen cross-linking affects the chondrocyte responses.
In this thesis, cell – tissue interactions in normal, aging and osteoarthritic articular cartilage were investigated. Chondrocytes were imaged by confocal laser scanning microscopy (CLSM) after osmotic or mechanical loading or without any loading to determine chondrocyte volume and shape. Reference techniques, such as biomechanical testing, polarized light microscopy (PLM), digital densitometry (DD), Fourier transform infrared microspectroscopy (FTIR) and high-performance liquid chromatography (HPLC), were utilized to define the function, structure and composition of samples. The samples were collected from normal healthy bovine lateral patellar groove (LPG) of the femur, bovine lateral aspect of the distal pole of
7 Huttu, Mari
Cell – tissue interactions in normal, aging and osteoarthritic articular cartilage Kuopio: University of Eastern Finland, 2017
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences 2017; 280 ISBN: 978-952-61-2589-3 (print)
ISSNL: 1798-5668 ISSN: 1798-5668
ISBN: 978-952-61-2590-9 (PDF) ISSN: 1798-5676 (PDF)
ABSTRACT
Articular cartilage is white, smooth, almost frictionless tissue without nerves or blood vessels covering ends of the long bones. It consists of one type of cells, chondrocytes, and extracellular matrix (ECM) comprising mainly tissue fluid, collagens and proteoglycans (PGs). The role of chondrocytes is to maintain the ECM.
Osteoarthritis (OA) is the most common joint disease in the world causing joint pain and decreased mobility of the joint. Age, gender, genetics, nutrition, obesity and abnormal joint loading are examples of risk factors of OA. Many early changes or symptoms, such as reduction of PG and disorganization of collagen fibrils, associated with OA are similar to changes in normal aging. On the other hand, aging also affects chondrocyte metabolism, and results in advanced glycation end products (AGEs) congregating the ECM. AGEs increase collagen cross-linking and ECM stiffness. Knowledge of the pathology OA and its progress is fairly well understood. However, at the cell and tissue level, many features are still unclear.
For example, which structural components and biomechanical properties of articular cartilage correlate together and participate in regulation of cell shape and volume in OA, are still unknown. It is also not known, how aging or induced collagen cross-linking affects the chondrocyte responses.
In this thesis, cell – tissue interactions in normal, aging and osteoarthritic articular cartilage were investigated. Chondrocytes were imaged by confocal laser scanning microscopy (CLSM) after osmotic or mechanical loading or without any loading to determine chondrocyte volume and shape. Reference techniques, such as biomechanical testing, polarized light microscopy (PLM), digital densitometry (DD), Fourier transform infrared microspectroscopy (FTIR) and high-performance liquid chromatography (HPLC), were utilized to define the function, structure and composition of samples. The samples were collected from normal healthy bovine lateral patellar groove (LPG) of the femur, bovine lateral aspect of the distal pole of
patellae and osteoarthritic human femoral heads. Half of the patellae samples were then ribose-treated for collagen cross-linking to simulate aging.
Superficial chondrocyte volume increase and volume recovery after hypo- osmotic challenge were observed not to be reliant on contents of immersion media or temperature. Chondrocyte volumes, in all media and temperature groups, first increased after hypotonic loading and then recovered back to control levels.
Simulated aging by in vitro glycation, was perceived to decrease chondrocyte deformation in the upper zone of articular cartilage during mechanical loading.
Mechanical forces were found to affect the deeper zone of articular cartilage by increasing chondrocyte deformation processes. In OA, the collagen fibril orientation and organization were found to be the most important ECM structural components to regulate chondrocyte volume in the superficial layer of articular cartilage. Depth- wise chondrocyte shape might be regulated more by PGs and collagen fibrils together. Alterations in collagen fibril orientation, organization and PGs and cell – tissue interactions in osteoarthritic tissue possibly affect the chondrocyte biosynthesis.
In conclusion, chondrocyte volume alterations after hypo-osmotic challenge are not dependent on immersion media or temperature. This is methodologically very important detail and underlines the significance of chondrocyte environment by comparing laboratory measurements to the real life. In simulated aging, alterations in the biomechanical responses of chondrocytes differ according to the zone of the articular cartilage. It is a noteworthy and important finding, how AGEs, one of the signs of aging in articular cartilage, affect the chondrocyte deformation processes divergently in the upper and the deeper zones of articular cartilage. Collagen fiber orientation angle chiefly regulate chondrocyte volume and depth-wise shape in osteoarthritic human hip cartilage. The results deepen the knowledge of osteoarthritic changes in cells and tissue in human cartilage.
National Library of Medicine Classification: QT 34.5, QU 55.3, WE 300, WE 348, WT 104 Medical Subject Headings: Cartilage, Articular; Chondrocytes; Collagen; Osteoarthritis, Aging; Osmotic Pressure; Stress, Mechanical; Biomechanical Phenomena; Cell Size; Cell Shape; Microscopy, Confocal
Yleinen suomalainen asiasanasto: nivelrusto; kollageenit; nivelrikko; ikääntyminen;
vanheneminen; biomekaniikka; solut; mikroskopia
ACKNOWLEDGEMENTS
The present study was carried out at the Department of Applied Physics, University of Eastern Finland, during the years 2009-2015.
First, I wish to thank my principal supervisor Professor Rami Korhonen, not only for his professional guidance and encouragement, but also for his patience and for allowing me the independence to learn things my way during my Ph.D. project.
I would also like to thank warmly my second supervisor Professor Mikko Lammi for his help.
I am very grateful to the official reviewers of my thesis, Holly Leddy, Ph.D. and Associate Professor Susanna Miettinen, Ph.D. for their constructive and valuable comments. I would also like to warmly thank Associate Professor Holly Shiels, Ph.D, for linguistic review.
I would like to express my deepest gratitude to all my co-authors for their significant contributions to the studies. I want to thank both Rami’s group and the BBC group for the enthusiastic atmosphere during this research work. I would like to acknowledge personnel from Institute of Biomedicine, Research services for their help on the sample preparation. I wish to thank also people from SibLabs for their guidance on the use of measurement devices and for sharing the coffee room.
Special thanks go to people who became my friends during my thesis project and my “old” friends outside university. Specifically, Virpi Tiitu, we have had many fruitful discussions inside and outside the university. Jari Leskinen, with whom I have had the pleasure of sharing a room for a couple of years. Ritva Savolainen and Virpi Miettinen, you always were ready to help in every kind of problem related or not related to the work. Your door has always been open for me.
Erja Kalliokoski, we have been friends over 30 years. Our nice little trips to Runni spa or to Tukholma have been really relaxing. I hope we have more nice little trips and other wonderful moments in the future! Elisa Nevalainen, you are my soul mate. Thank you every crazy memories, such as Independence Day dinner in the lab! Niina Tani, we have spent hours and hours of our free time with the horses. We have cried together and laughed together, that’s life with the horses! Thank you for your friendship! Johanna Sokka, my personal veterinarian, we have had good conversations early in the mornings and during night times. We have also had memorable parties. Thank you for being there! Emmi Väisänen, the second
“mother” to my wonderful horse Ritzferal. Thank you for spectacular riding and care of my special Horse and for sharing special moments with dressage. Anna Jauhiainen, almost the first person I got to know in Kuopio. Thank you for your friendship and for being a mentor in the horse stable things!
I wish to express my dearest thanks to my beloved husband Timo for his endless love, understanding and for helping in everything: cleaning the house, shoeing the horses, caring of the children, cats and fowls… I thank my wonderful children, Siiri and Saimi for their energy, joyfulness and laugh. Finally, I am grateful to my
9
ACKNOWLEDGEMENTS
The present study was carried out at the Department of Applied Physics, University of Eastern Finland, during the years 2009-2015.
First, I wish to thank my principal supervisor Professor Rami Korhonen, not only for his professional guidance and encouragement, but also for his patience and for allowing me the independence to learn things my way during my Ph.D. project.
I would also like to thank warmly my second supervisor Professor Mikko Lammi for his help.
I am very grateful to the official reviewers of my thesis, Holly Leddy, Ph.D. and Associate Professor Susanna Miettinen, Ph.D. for their constructive and valuable comments. I would also like to warmly thank Associate Professor Holly Shiels, Ph.D, for linguistic review.
I would like to express my deepest gratitude to all my co-authors for their significant contributions to the studies. I want to thank both Rami’s group and the BBC group for the enthusiastic atmosphere during this research work. I would like to acknowledge personnel from Institute of Biomedicine, Research services for their help on the sample preparation. I wish to thank also people from SibLabs for their guidance on the use of measurement devices and for sharing the coffee room.
Special thanks go to people who became my friends during my thesis project and my “old” friends outside university. Specifically, Virpi Tiitu, we have had many fruitful discussions inside and outside the university. Jari Leskinen, with whom I have had the pleasure of sharing a room for a couple of years. Ritva Savolainen and Virpi Miettinen, you always were ready to help in every kind of problem related or not related to the work. Your door has always been open for me.
Erja Kalliokoski, we have been friends over 30 years. Our nice little trips to Runni spa or to Tukholma have been really relaxing. I hope we have more nice little trips and other wonderful moments in the future! Elisa Nevalainen, you are my soul mate. Thank you every crazy memories, such as Independence Day dinner in the lab! Niina Tani, we have spent hours and hours of our free time with the horses. We have cried together and laughed together, that’s life with the horses! Thank you for your friendship! Johanna Sokka, my personal veterinarian, we have had good conversations early in the mornings and during night times. We have also had memorable parties. Thank you for being there! Emmi Väisänen, the second
“mother” to my wonderful horse Ritzferal. Thank you for spectacular riding and care of my special Horse and for sharing special moments with dressage. Anna Jauhiainen, almost the first person I got to know in Kuopio. Thank you for your friendship and for being a mentor in the horse stable things!
I wish to express my dearest thanks to my beloved husband Timo for his endless love, understanding and for helping in everything: cleaning the house, shoeing the horses, caring of the children, cats and fowls… I thank my wonderful children, Siiri and Saimi for their energy, joyfulness and laugh. Finally, I am grateful to my
parents, Ritva and Markku, for their continuous support and love throughout my life.
Finally, I want to acknowledge Sigrid Juselius Foundation, Finnish Cultural Foundation, Magnus Ehrnrooth Foundation, Orion Pharma Research Foundation, Doctoral Programme in Medical Physics and Engineering of University of Eastern Finland, Kuopio University Hospital, Aleksanteri Mikkonen Foundation, National Doctoral Programme of Muculoskeletal Disorders and Biomaterials (TBDP), Atria lihakunta Oyj, Kuopio.
Kuopio, 18th August 2017 Mari Huttu
LIST OF ABBREVIATIONS
AC Articular cartilage
ADAMTS A disintegrin and metalloproteinase with thrombospondin motif AGEs Advanced glycation end products
BMPs Bone morphogenetic proteins
CDMPs Cartilage-derived morphogenic proteins CLSM Confocal laser scanning microscopy CTGF Connective-tissue growth factor DD Digital densitometry
DMEM Dulbecco´s modified Eagle´s medium DZ Deep zone of cartilage
ECM Extracellular matrix FCD Fixed charge density FGF Fibroblast growth factor
FTIR Fourier transform infrared microspectroscopy GAG Glycosaminoglycan
HGF Hepatocyte growth factor HP Hydroxylysyl pyridinoline
HPLC High-performance liquid chromatography IGFs Insulin-like growth factors
LM Light microscopy LP Lysyl pyridinoline
LPG Lateral patellar groove of femur MMPs Matrix metalloproteinases MZ Middle zone of cartilage OA Osteoarthritis
PBS Phosphate-buffered saline PCM Pericellular matrix Pent Pentosidine
PG Proteoglycan
PI Propidium iodide
PLM Polarized light microscopy RVD Regulatory volume decrease TNF-α Tumor necrosis factor-α TGF-β Transforming growth factor-β UZ Upper zone of cartilage
11
LIST OF ABBREVIATIONS
AC Articular cartilage
ADAMTS A disintegrin and metalloproteinase with thrombospondin motif AGEs Advanced glycation end products
BMPs Bone morphogenetic proteins
CDMPs Cartilage-derived morphogenic proteins CLSM Confocal laser scanning microscopy CTGF Connective-tissue growth factor DD Digital densitometry
DMEM Dulbecco´s modified Eagle´s medium DZ Deep zone of cartilage
ECM Extracellular matrix FCD Fixed charge density FGF Fibroblast growth factor
FTIR Fourier transform infrared microspectroscopy GAG Glycosaminoglycan
HGF Hepatocyte growth factor HP Hydroxylysyl pyridinoline
HPLC High-performance liquid chromatography IGFs Insulin-like growth factors
LM Light microscopy LP Lysyl pyridinoline
LPG Lateral patellar groove of femur MMPs Matrix metalloproteinases MZ Middle zone of cartilage OA Osteoarthritis
PBS Phosphate-buffered saline PCM Pericellular matrix Pent Pentosidine
PG Proteoglycan
PI Propidium iodide
PLM Polarized light microscopy RVD Regulatory volume decrease TNF-α Tumor necrosis factor-α TGF-β Transforming growth factor-β UZ Upper zone of cartilage
LIST OF SYMBOLS
𝜙𝜙int Internal osmotic coefficient 𝜙𝜙ext External osmotic coefficient 𝛾𝛾int Internal activity coefficient 𝛾𝛾ext External activity coefficient R Molar gas constant
T Absolute temperature 𝑐𝑐F Fixed charge density 𝑐𝑐ext External salt concentration Eeq Equilibrium modulus E0 Initial modulus
Eε Strain-dependent modulus
13
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on data presented in the following articles, referrred to by the Roman Numerals I-III.
I Huttu M R J, Turunen S M, Sokolinski V, Tiitu V, Lammi M J and Korhonen R K. (2012). Effects of medium and temperature on cellular responses in the superficial zone of hypo-osmotically challenged articular cartilage. Journal of Functional Biomaterials, 3 (3): 544-555.
II Fick J M*, Huttu M R J*, Lammi M J and Korhonen R K. (2014). In vitro glycation of articular cartilage alters the biomechanical response of
chondrocytes in a depth-dependent manner. Osteoarthritis and Cartilage, 22:
1410-1418.
III Huttu M R J, Puhakka J, Mäkelä J T A, Tiitu V, Saarakkala S, Konttinen Y T, Kiviranta I and Korhonen R K. (2014). Cell – tissue interactions in
osteoarthritic human hip joint articular cartilage. Connective Tissue Research, 55 (4): 282-291.
The original articles have been reproduced with the permission of the copyright holders.
*Equal contribution
AUTRHOR’S CONTRIBUTION
The publications in this dissertation are original research papers addressing the cellular responses to loading and cell – tissue interactions in aged and osteoarthritic articular cartilage. The author has contributed to planning of each study.
In study I, the author has done all confocal microscopy measurements, and con- tributed to their accomplishment in studies II and III. The author has done all the cell volume analysis in all studies. The author has carried out all polarized light microscopy and Fourier transform infrared microspectroscopy measurements and analyses in study II. The author has also conducted all light microscopy, polarized light microscopy and digital densitometry measurements and analyses in study III.
The author has participated in each method and analysis presented in publications, excluding biochemical analysis. Furthermore, the author has carried out all the statistical analyses in study III, and participated to those in studies I and II. The author was the main writer in studies I and III, and there was an equal contribution with the first author in study II.
15
AUTRHOR’S CONTRIBUTION
The publications in this dissertation are original research papers addressing the cellular responses to loading and cell – tissue interactions in aged and osteoarthritic articular cartilage. The author has contributed to planning of each study.
In study I, the author has done all confocal microscopy measurements, and con- tributed to their accomplishment in studies II and III. The author has done all the cell volume analysis in all studies. The author has carried out all polarized light microscopy and Fourier transform infrared microspectroscopy measurements and analyses in study II. The author has also conducted all light microscopy, polarized light microscopy and digital densitometry measurements and analyses in study III.
The author has participated in each method and analysis presented in publications, excluding biochemical analysis. Furthermore, the author has carried out all the statistical analyses in study III, and participated to those in studies I and II. The author was the main writer in studies I and III, and there was an equal contribution with the first author in study II.
CONTENTS
ABSTRACT ... 7
ACKNOWLEDGEMENTS ... 9
1 INTRODUCTION ... 19
2 ARTICULAR CARTILAGE ... 21
2.1 Structure and function ...21
2.1.1 Chondrocytes ...22
2.1.2 Interstitial fluid ...23
2.1.3 Collagen ...23
2.1.4 Proteoglycans ...23
2.2 Aging and osteoarthritis ...26
3 CHONDROCYTE RESPONSES IN ARTICULAR CARTILAGE ... 28
3.1 Chondrocyte responses to osmotic load ...28
3.2 Chondrocyte responses to mechanical load ...31
3.3 Cell – tissue interactions ...31
3.3.1 Effects of aging ...32
3.3.2 Effects of osteoarthritis...33
4 AIMS OF THE STUDY ... 35
5 MATERIALS AND METHODS ... 36
5.1 Sample preparation ...37
5.2 Confocal laser scanning microscopy ...38
5.2.1 Osmotic loading ...39
5.2.2 Mechanical loading ...41
5.2.3 Osteoarthritic samples ...41
5.3 Light microscopy...42
5.4 Polarized light microscopy ...42
5.5 Digital densitometry ...42
5.6 FTIR microspectroscopy ...44
5.7 Biomechanical testing ...44
5.8 Biochemical analysis ...45
5.9 Statistical analysis ...45
6 RESULTS ... 47
6.1 Chondrocyte response to osmotic loading in normal cartilage ...47
6.1.1 Resting volume ...47
6.1.2 Effects of media and temperature ...47
6.2 Chondrocyte response to mechanical loading in normal cartilage ...49
6.2.1 Chondrocyte volume and morphology ... 49
6.2.2 Local axial and transverse strains of the tissue ... 50
6.2.3 Structural and mechanical analysis of the cartilage ... 51
6.3 Cell – tissue interactions in osteoarthritic cartilage ... 53
6.3.1 Chondrocyte volume and morphology ... 53
6.3.2 Effects of osteoarthrits ... 53
7 DISCUSSION ... 56
7.1 Chondrocyte response to osmotic loading in normal cartilage in two different media and temperature ... 59
7.2 Chondrocyte response to mechanical loading and cell – tissue interactions in aging cartilage ... 60
7.3 Cell – tissue interactions in osteoarthritic cartilage ... 61
7.4 Limitations ... 63
8 SUMMARY AND CONCLUSIONS ... 64
BIBLIOGRAPHY ... 65
17
6.2.1 Chondrocyte volume and morphology ... 49
6.2.2 Local axial and transverse strains of the tissue ... 50
6.2.3 Structural and mechanical analysis of the cartilage ... 51
6.3 Cell – tissue interactions in osteoarthritic cartilage ... 53
6.3.1 Chondrocyte volume and morphology ... 53
6.3.2 Effects of osteoarthrits ... 53
7 DISCUSSION ... 56
7.1 Chondrocyte response to osmotic loading in normal cartilage in two different media and temperature ... 59
7.2 Chondrocyte response to mechanical loading and cell – tissue interactions in aging cartilage ... 60
7.3 Cell – tissue interactions in osteoarthritic cartilage ... 61
7.4 Limitations ... 63
8 SUMMARY AND CONCLUSIONS ... 64
BIBLIOGRAPHY ... 65
1 INTRODUCTION
Articular cartilage is a hyaline cartilage, which covers the subchondral bone ends on the articulating surfaces (Mow et al., 1992). There are no blood vessels, lymphatic vessels or nerves in a hyaline cartilage. It consists of extracellular matrix (ECM) and one type of cell, chondrocytes. The ECM is composed mainly from collagen fibrils, proteoglycans (PGs) and tissue fluid. The collagen and PG content and collagen fibril orientation vary depending on depth from the surface of articular cartilage (Buckwalter and Mankin, 1997a; Mow et al., 1992; Poole et al., 2001). Also the shape and activity of chondrocytes differ at different depths of the tissue. The collagen fibrils contribute primarily to the tensile stiffness (Buckwalter and Mankin, 1997a; Martel-Pelletier et al., 2008) and PGs to the compressive stiffness of articular cartilage (Hascall and Hascall, 1981; Mow et al., 1992).
Chondrocytes nurture the ECM by synthesizing and catabolizing its components (Buckwalter et al., 1994; Buckwalter and Mankin, 1997a; Lin et al., 2006).
Osteoarthritis (OA) is the most common degenerative joint disease across the world (Arden and Nevitt, 2006). Etiology of OA is not clear, but assumed risk factors are for example age, gender, ethnicity, nutrition, obesity and injuries. OA affects the structure, composition and function of articular cartilage. Reduction of especially superficial PGs and altered collagen fibril organization are the first structural changes in the early stages of OA (Bi et al., 2006; Buckwalter and Mankin, 1997b). These changes might also be similar in normal aging (Freeman and Meachim, 1979). During progression of OA the water content increases (Bank et al., 2000; Freeman and Meachim, 1979), and the collagen and PG contents decrease gradually in articular cartilage (Bi et al., 2006; Saarakkala et al., 2010). Thus, the tissue permeability increases while the compressive and tensile stiffnesses decrease (Buckwalter and Mankin, 1997b; Roberts et al., 1986). However, during normal aging process the stiffness of cartilage tissue may increase (Bank et al., 1998; Chen et al., 2002; Eyre et al., 1988; Verzijl et al., 2000b, 2000a, 2002). This is apparently a consequence of collagen cross-linking. Aging affects also viscoelastic properties of the pericellular matrix (PCM) (Duan et al., 2012), chondrocyte metabolism (Bank et al., 1998; DeGroot et al., 2001, 1999; Hügle et al., 2012; Jørgensen et al., 2017; Li et al., 2013; Lotz and Loeser, 2012; Verzijl et al., 2003; Vos et al., 2012; Wilkins et al., 2000), metabolic pathways and advanced glycation end products (AGEs) gathering in the ECM (Chen et al., 2002; DeGroot et al., 1999, 2001; Duan et al., 2012; Verzijl et al., 2000b, 2000a, 2002; Vos et al., 2012). Age-dependent collagen cross-linking might affect the function of articular cartilage and, thus, advance degeneration of the tissue leading to OA (Bader et al., 2011; Hügle et al., 2012; Li et al., 2013; Lotz and Loeser, 2012; Sun, 2010; Verzijl et al., 2003; Wilkins et al., 2000).
In this thesis, the cellular, structural and/or functional characteristics of articular cartilage were studied during hypo-osmotic challenge in different media and
19
1 INTRODUCTION
Articular cartilage is a hyaline cartilage, which covers the subchondral bone ends on the articulating surfaces (Mow et al., 1992). There are no blood vessels, lymphatic vessels or nerves in a hyaline cartilage. It consists of extracellular matrix (ECM) and one type of cell, chondrocytes. The ECM is composed mainly from collagen fibrils, proteoglycans (PGs) and tissue fluid. The collagen and PG content and collagen fibril orientation vary depending on depth from the surface of articular cartilage (Buckwalter and Mankin, 1997a; Mow et al., 1992; Poole et al., 2001). Also the shape and activity of chondrocytes differ at different depths of the tissue. The collagen fibrils contribute primarily to the tensile stiffness (Buckwalter and Mankin, 1997a; Martel-Pelletier et al., 2008) and PGs to the compressive stiffness of articular cartilage (Hascall and Hascall, 1981; Mow et al., 1992).
Chondrocytes nurture the ECM by synthesizing and catabolizing its components (Buckwalter et al., 1994; Buckwalter and Mankin, 1997a; Lin et al., 2006).
Osteoarthritis (OA) is the most common degenerative joint disease across the world (Arden and Nevitt, 2006). Etiology of OA is not clear, but assumed risk factors are for example age, gender, ethnicity, nutrition, obesity and injuries. OA affects the structure, composition and function of articular cartilage. Reduction of especially superficial PGs and altered collagen fibril organization are the first structural changes in the early stages of OA (Bi et al., 2006; Buckwalter and Mankin, 1997b). These changes might also be similar in normal aging (Freeman and Meachim, 1979). During progression of OA the water content increases (Bank et al., 2000; Freeman and Meachim, 1979), and the collagen and PG contents decrease gradually in articular cartilage (Bi et al., 2006; Saarakkala et al., 2010). Thus, the tissue permeability increases while the compressive and tensile stiffnesses decrease (Buckwalter and Mankin, 1997b; Roberts et al., 1986). However, during normal aging process the stiffness of cartilage tissue may increase (Bank et al., 1998; Chen et al., 2002; Eyre et al., 1988; Verzijl et al., 2000b, 2000a, 2002). This is apparently a consequence of collagen cross-linking. Aging affects also viscoelastic properties of the pericellular matrix (PCM) (Duan et al., 2012), chondrocyte metabolism (Bank et al., 1998; DeGroot et al., 2001, 1999; Hügle et al., 2012; Jørgensen et al., 2017; Li et al., 2013; Lotz and Loeser, 2012; Verzijl et al., 2003; Vos et al., 2012; Wilkins et al., 2000), metabolic pathways and advanced glycation end products (AGEs) gathering in the ECM (Chen et al., 2002; DeGroot et al., 1999, 2001; Duan et al., 2012; Verzijl et al., 2000b, 2000a, 2002; Vos et al., 2012). Age-dependent collagen cross-linking might affect the function of articular cartilage and, thus, advance degeneration of the tissue leading to OA (Bader et al., 2011; Hügle et al., 2012; Li et al., 2013; Lotz and Loeser, 2012; Sun, 2010; Verzijl et al., 2003; Wilkins et al., 2000).
In this thesis, the cellular, structural and/or functional characteristics of articular cartilage were studied during hypo-osmotic challenge in different media and
temperature, both in aging and in OA cartilages. Specifically, 1) the cellular responses of articular cartilage were studied under hypo-osmotic challenge in different immersion media and at different temperatures, 2) the cellular responses, structural and functional alterations of simulated aging articular cartilage were investigated under compression, and 3) the structural, functional and cellular interactions of articular cartilage during the progression of OA were evaluated from human hip joint. Chondrocytes were imaged by confocal microscopy in normal, simulated aging and osteoarthritic articular cartilage. Fourier transform infrared (FTIR) microspectroscopy, polarized light microscopy (PLM), digital densitometry (DD) and mechanical testing were used to study the structure, composition and function of articular cartilage samples.
The results of the present study offer methodologically and translationally important knowledge of the cartilage by comparing laboratory circumstances to the real environment of chondrocytes. This study also offers an interpretation on how aging affects the chondrocyte deformation processes. Further, the results of this study deepen the knowledge of changes in cell and tissue in human osteoarthritic cartilage during progression of OA
2 ARTICULAR CARTILAGE
2.1 STRUCTURE AND FUNCTION
Articular cartilage is hyaline cartilage covering the ends of the long bones (Mow et al., 1992). It is an aneural and avascular connective tissue, which consists of a fluid phase containing water and electrolytes and a solid phase containing chondrocytes, collagens, PGs and glycoproteins.
Articular cartilage can be typically divided into four histologically different zones: superficial, middle, deep and calcified zone (Figure 2.1)(Buckwalter, 1983;
Poole et al., 2001). The zones differ with respect to chondrocytes size, shape, metabolic activity, water content, collagen content and orientation, and PG content.
Figure 2.1: Schematic picture of the histological zones of articular cartilage. The first cartilage plug shows chondrocyte shape and organization and the second plug shows collagen fiber orientation in different histological zones of articular cartilage.
Superficial zone
Middle zone
Deep zone Articular surface
Tidemark Bone
21
2 ARTICULAR CARTILAGE
2.1 STRUCTURE AND FUNCTION
Articular cartilage is hyaline cartilage covering the ends of the long bones (Mow et al., 1992). It is an aneural and avascular connective tissue, which consists of a fluid phase containing water and electrolytes and a solid phase containing chondrocytes, collagens, PGs and glycoproteins.
Articular cartilage can be typically divided into four histologically different zones: superficial, middle, deep and calcified zone (Figure 2.1)(Buckwalter, 1983;
Poole et al., 2001). The zones differ with respect to chondrocytes size, shape, metabolic activity, water content, collagen content and orientation, and PG content.
Figure 2.1: Schematic picture of the histological zones of articular cartilage. The first cartilage plug shows chondrocyte shape and organization and the second plug shows collagen fiber orientation in different histological zones of articular cartilage.
Superficial zone
Middle zone
Deep zone Articular surface
Tidemark Bone
2.1.1 Chondrocytes
Articular cartilage contains a unique type of cells: chondrocytes. A mean diameter of chondrocytes is 13 µm (Hunziker et al., 2002; Lin et al., 2006). The chondrocyte density is only approximately 2-10% of the total articular cartilage volume (Lin et al., 2006; Poole et al., 2001), depending on the maturation of the individual and the species (Kamisan et al., 2013; Stockwell, 1967). The role of chondrocytes in articular cartilage is to maintain the stable ECM by biosynthesis and catabolism (Hunziker et al., 2002; Lin et al., 2006; Urban, 1994).
In the superficial zone, the chondrocytes are flattened, elongated and lined up parallel to the cartilage surface (Figure 2.1) (Buckwalter and Mankin, 1997a; Poole et al., 2001). In the middle zone, they are more spherical and the cell density is lower than in the superficial zone (Poole et al., 2001). The deep zone chondrocytes are spheroidal and columnar, orienting vertical to the cartilage surface (Newman, 1998). The cell density decreases with depth from the surface (Jadin, 2005).
The chondrocytes in all the zones contain round or oval nucleus, a pair of centrioles, endoplasmic reticulum, well-developed Golgi apparatus, mitochondria, lipids and glycogen in the cytoplasm (Bloom, 1975). The microtubules, the intermediate filaments and the microfilaments, which contain proteins such as tubulin, vimentin and actin, respectively, are constituents of the cytoskeleton of chondrocytes (Trickey et al., 2004). The microfilaments and the intermediate filaments mainly determine the viscoelastic behaviour of the chondrocytes.
Additionally, the microtubules and the microfilaments are known to be sensitive to temperature (Hall, 1995). Lower temperatures than physiological temperature, 37°C, can change the assembly, and, thus the stiffness of the microfilament network (Tan et al., 2008). Thus, the cytoskeleton plays an important role to define the biomechanical properties of chondrocytes (Tan et al., 2008; Trickey et al., 2004).
The ECM of articular cartilage is maintained by the chondrocytes (Buckwalter et al., 1994; Buckwalter and Mankin, 1997a; Lin et al., 2006). Thus, they are responsible for the anabolic and the catabolic activities of the ECM macromolecules. Mechanical loading substantially regulates the metabolic activity of chondrocytes. The loading frequency and amplitude strongly affect chondrocyte anabolic and catabolic activities (Urban, 1994). In addition, the state of the ECM has an important impact on chondrocyte function (Buckwalter and Mankin, 1997a). The rate of chondrocyte metabolic activity and function is linked to the age of an individual. In growing individuals, the chondrocytes proliferate, divide and synthesize new ECM, effectively secreting and remodeling the articular cartilage. In skeletally mature individuals, expanding new cartilage is rare and an important anabolic activity of the chondrocytes is to replace the catabolized ECM macromolecules (Buckwalter and Mankin, 1997a; Newman, 1998).
2.1.2 Interstitial fluid
Interstitial fluid, composed of mainly water and dissolved electrolytes, comprises 60-85% of the articular cartilage wet weight (Martel-Pelletier et al., 2008; Mow et al., 1992). The amount of the interstitial fluid is the highest in the superficial zone and decreases gradually towards the deep zone (Maroudas and Venn, 1977; Martel- Pelletier et al., 2008; Mow et al., 1992).
The interstitial fluid flow supplies nutrients to the chondrocytes and removes metabolites away and together with synovial fluid, ensures lubrication of the ar- ticular cartilage surface (Mow et al., 1984, 1992). Moreover, the interstitial fluid plays an important role in determining the mechanical properties of cartilage under dynamic and static loading conditions (Kempson, G. E., 1973; Maroudas, A., 1973;
Mow et al., 1984, 1992).
2.1.3 Collagen
About 15-22% of the wet weight or 50-80% of the dry weight of the articular carti- lage is made up of collagen (Mow et al., 1992). Several genetically different collagen molecules, types I, II, III, VI, IX, X, XI, XII and XIV, have been found in articular cartilage (Eyre, 2002). However, at least 90-95% of the collagen in articular cartilage is type II collagen (Buckwalter and Mankin, 1997a; Eyre, 2002; Mow et al., 1992).
Collagen molecules are triple-helical protein structures containing three α poly- peptide chains (Figure 2.2a) (Buckwalter and Mankin, 1997a; Eyre, 2002; Mow et al., 1992; Prockop and Kivirikko, 1995). Hydrogen bonds and water bridges stabilize the triple-helix structure (Prockop and Kivirikko, 1995). Thus, the shape and form, as well as the tensile stiffness and strength, of the articular cartilage tissue are pro- vided by the collagen fibril network (Buckwalter and Mankin, 1997a; Martel- Pelletier et al., 2008).
Diameter of collagen fibrils is thinnest, about 20 nm, in the superficial zone and increases to 70-120 nm towards the deep zone (Eyre, 2002; Martel-Pelletier et al., 2008). Collagen fibrils are organized parallel to the joint surface in the superficial zone (Figure 1) (Buckwalter and Mankin, 1997a; Eyre, 2002; Newman, 1998; Poole et al., 2001). In the middle zone, collagen fibrils are orientated more randomly, and in the deep zone, they are vertical to the articular cartilage surface (Figure 2.1).
2.1.4 Proteoglycans
Proteoglycans account for approximately 4-7% of articular cartilage wet weight (Mow et al., 1992). They contain a protein core to which glycosaminoglycan (GAG) chains are covalently linked (Figure 2.2b) (Buckwalter and Mankin, 1997a; Hascall and Hascall, 1981; Mow et al., 1992). The protein core comprise only about 5-10% of
23 2.1.2 Interstitial fluid
Interstitial fluid, composed of mainly water and dissolved electrolytes, comprises 60-85% of the articular cartilage wet weight (Martel-Pelletier et al., 2008; Mow et al., 1992). The amount of the interstitial fluid is the highest in the superficial zone and decreases gradually towards the deep zone (Maroudas and Venn, 1977; Martel- Pelletier et al., 2008; Mow et al., 1992).
The interstitial fluid flow supplies nutrients to the chondrocytes and removes metabolites away and together with synovial fluid, ensures lubrication of the ar- ticular cartilage surface (Mow et al., 1984, 1992). Moreover, the interstitial fluid plays an important role in determining the mechanical properties of cartilage under dynamic and static loading conditions (Kempson, G. E., 1973; Maroudas, A., 1973;
Mow et al., 1984, 1992).
2.1.3 Collagen
About 15-22% of the wet weight or 50-80% of the dry weight of the articular carti- lage is made up of collagen (Mow et al., 1992). Several genetically different collagen molecules, types I, II, III, VI, IX, X, XI, XII and XIV, have been found in articular cartilage (Eyre, 2002). However, at least 90-95% of the collagen in articular cartilage is type II collagen (Buckwalter and Mankin, 1997a; Eyre, 2002; Mow et al., 1992).
Collagen molecules are triple-helical protein structures containing three α poly- peptide chains (Figure 2.2a) (Buckwalter and Mankin, 1997a; Eyre, 2002; Mow et al., 1992; Prockop and Kivirikko, 1995). Hydrogen bonds and water bridges stabilize the triple-helix structure (Prockop and Kivirikko, 1995). Thus, the shape and form, as well as the tensile stiffness and strength, of the articular cartilage tissue are pro- vided by the collagen fibril network (Buckwalter and Mankin, 1997a; Martel- Pelletier et al., 2008).
Diameter of collagen fibrils is thinnest, about 20 nm, in the superficial zone and increases to 70-120 nm towards the deep zone (Eyre, 2002; Martel-Pelletier et al., 2008). Collagen fibrils are organized parallel to the joint surface in the superficial zone (Figure 1) (Buckwalter and Mankin, 1997a; Eyre, 2002; Newman, 1998; Poole et al., 2001). In the middle zone, collagen fibrils are orientated more randomly, and in the deep zone, they are vertical to the articular cartilage surface (Figure 2.1).
2.1.4 Proteoglycans
Proteoglycans account for approximately 4-7% of articular cartilage wet weight (Mow et al., 1992). They contain a protein core to which glycosaminoglycan (GAG) chains are covalently linked (Figure 2.2b) (Buckwalter and Mankin, 1997a; Hascall and Hascall, 1981; Mow et al., 1992). The protein core comprise only about 5-10% of
the entire proteoglycan molecule mass. GAG chains, composed mainly of chon- droitin sulfate and keratan sulfate, occupy most of the proteoglycan molecular mass (Hascall and Hascall, 1981). GAG chains are negatively charged, whereupon they repel other anionic molecules and bind cations and water (Buckwalter and Mankin, 1997a; Mow et al., 1992).
The PGs in articular cartilage are mainly the large aggregating type, 50-85% ag- grecan, and the large non-aggregating type 10-40% (Martel-Pelletier et al., 2008;
Mow et al., 1992). Additionally, small PGs, about 3%, like decorin, biglycan, fibro- modulin and lumican, are located in articular cartilage (Buckwalter and Mankin, 1997a).
Proteoglycan aggregates comprise many aggrecans non-covalently linked with hyaluronan and small non-collagenous proteins called link proteins (Figure 2.2b) (Buckwalter and Mankin, 1997a; Mow et al., 1992). Aggregate formation is im- portant to attach the PGs within the ECM, to stabilize the PGs during compression and to compose stable relationship between the PGs and the collagens. In the super- ficial zone of articular cartilage, the aggrecan content is the lowest, but decorin and biglycan contents are the highest (Poole et al., 2001). The aggrecan content is high- est in the deep zone of the articular cartilage.
Negatively charged PGs create a swelling pressure on the cartilage tissue (Maroudas and Bannon, 1981). The collagen fibril network resists this pressure.
Thus, the PGs, mainly aggregans, affect the compressive stiffness of the articular cartilage by providing a hydrated, viscous, load absorbing gel (Hascall and Hascall, 1981; Mow et al., 1992).
Figure 2.2: Schematic structure of the collagen molecule (a) and the aggrecan PG molecule (b).
25 Figure 2.2: Schematic structure of the collagen molecule (a) and the aggrecan PG molecule (b).
2.2 AGING AND OSTEOARTHRITIS
The normal aging process increases the stiffness of the ECM of the articular carti- lage (Bank et al., 1998; Chen et al., 2002; Eyre et al., 1988; Verzijl et al., 2000b, 2000a, 2002) and the chondrocytes (Chahine et al., 2013; Duan et al., 2012). Aging reduces also the viscoelastic properties of the PCM (Duan et al., 2012). Additionally, aging affects chondrocyte metabolism and modifies the metabolic pathways (Bank et al., 1998; DeGroot et al., 1999, 2001; Hügle et al., 2012; Jørgensen et al., 2017; Li et al., 2013; Lotz and Loeser, 2012; Verzijl et al., 2003; Vos et al., 2012; Wilkins et al., 2000).
During aging, AGEs gather to the ECM and increase, for example, the collagen cross-linking (Chen et al., 2002; DeGroot et al., 1999, 2001; Duan et al., 2012; Verzijl et al., 2000b, 2000a, 2002; Vos et al., 2012). The collagen cross-linking has been re- ported to influence the function of the articular cartilage, possibly leading to tissue degeneration and OA as a consequence of stiffer tissue (Bader et al., 2011; Hügle et al., 2012; Jørgensen et al., 2017; Li et al., 2013; Lotz and Loeser, 2012; Sun, 2010; Ver- zijl et al., 2003; Wilkins et al., 2000). As a significant result of the normal aging pro- cess, tissue may become more prone to fractures (Chen et al., 2002; Verzijl et al., 2000a, 2002).
Osteoarthritis is worldwide the most common joint disease, which primarily causes joint pain and decreased mobility of the joint (Arden and Nevitt, 2006). Ra- diographic signs of OA occur in about 80% of people aged over 75 years in the western populations. In humans, OA affects most typically small joints in the hands and large weight-bearing joints such as hip and knee (Hunter and Felson, 2006).
The etiology of OA is not fully understood, but several risk factors, systemic and local mechanical, are clearly understood (Arden and Nevitt, 2006). The systemic risk factors for OA are age, gender ethnicity and race, genetics and nutrition (Arden and Nevitt, 2006; Johnson and Hunter, 2014). The local mechanical risk factors are obesity, acute joint injuries, repetitive or abnormal joint loading and muscle strength or weakness. Many early changes traditionally associated with OA are similar to changes in normal aging process (Martel-Pelletier et al., 2008).
The degenerative symptoms of OA focus on the structure, composition and function of the articular cartilage. One of the earliest signs of OA is a reduction in the superficial PG content (Bi et al., 2006; Buckwalter and Mankin, 1997b; Guilak et al., 1994). Although the superficial fibrillation and disruption of the collagen net- work has reported to occur in the early stages of OA (Bi et al., 2006; Buckwalter and Mankin, 1997b; Panula et al., 1998), it happens also during aging (Freeman and Meachim, 1979). However, collagen cross-linking may occur during aging and in- crease the stiffness of the articular cartilage (Chen et al., 2002). During the progres- sion of OA, the water content of the articular cartilage increases and cartilage first swells, but later becomes thinner (Bank et al., 2000; Freeman and Meachim, 1979).
Additionally, the collagen content of the articular cartilage decreases in later stages of OA (Bi et al., 2006; Saarakkala et al., 2010). The above-mentioned changes lead to
increased permeability and decreased compressive stiffness of the articular carti- lage (Buckwalter and Mankin, 1997b; Roberts et al., 1986). These changes are very site-specific in the joint (Mäkelä et al., 2015). Also, the environment of the chondro- cytes becomes hypotonic (Bush and Hall, 2005; Maroudas and Venn, 1977). Thus, chondrocyte volume increases in the degenerated articular cartilage (Bush and Hall, 2003). Nevertheless, chondrocyte morphology, defined as aspect ratio, height/width, seems not to change until in the more advanced stages of OA (Korhonen et al., 2011).
27 increased permeability and decreased compressive stiffness of the articular carti- lage (Buckwalter and Mankin, 1997b; Roberts et al., 1986). These changes are very site-specific in the joint (Mäkelä et al., 2015). Also, the environment of the chondro- cytes becomes hypotonic (Bush and Hall, 2005; Maroudas and Venn, 1977). Thus, chondrocyte volume increases in the degenerated articular cartilage (Bush and Hall, 2003). Nevertheless, chondrocyte morphology, defined as aspect ratio, height/width, seems not to change until in the more advanced stages of OA (Korhonen et al., 2011).
3 CHONDROCYTE RESPONSES IN ARTICU- LAR CARTILAGE
The structure and composition of the articular cartilage alters the chondrocyte re- sponse to both osmotic (Bush and Hall, 2003; Turunen et al., 2012b; Urban et al., 1993) and mechanical load (Korhonen and Herzog, 2008; Turunen et al., 2013).
Chondrocyte volume change and regulation are important phenomena to cartilage physiology and vital to the chondrocytes (Lewis et al., 2011). It occurs in several different situations. For example, with aging and exercise, the chondrocyte volume increases. During osmotic loading it increases rapidly and, thereafter, a regulatory volume decrease (RVD) response occurs leading to chondrocyte volume recovery in normal cartilage (Bush and Hall, 2001a; Turunen et al., 2012a). In OA, osmolarity of the cartilage is reported to decrease (Maroudas and Venn, 1977), and RVD is per- haps disturbed as a consequence of the ECM metabolic changes (Bush and Hall, 2003). During compressive loading the chondrocytes flatten, which induces the cell membrane stretching, and the chondrocyte volume increase. After this phenome- non, the chondrocyte volume reduces actively in normal articular cartilage as a result of the volume regulatory mechanisms. A decline of these mechanisms per- haps affect the progression of OA (Bush and Hall, 2003). Indeed, during compres- sive loading of osteoarthritic cartilage the chondrocyte volume does not reduce, but rather increases (Han et al., 2010; Turunen et al., 2013). Several changes in healthy aging cartilage are opposed to changes in progression of OA (Grushko et al., 1989).
For example, the fixed charged density (FCD) and osmotic pressure increase.
3.1 CHONDROCYTE RESPONSES TO OSMOTIC LOAD
PGs of the articular cartilage ECM are negatively charged and pull in positively- charged ions, mainly Na+ (Maroudas, A., 1973). Difference in ion concentration between the tissue and its environment forms a swelling pressure. The fluid starts to flow and ion concentration aspires toward equilibrium by diffusion. This phe- nomenon is called Donnan swelling pressure (Wilson et al., 2005):
∆𝜋𝜋 = 𝜙𝜙int𝑅𝑅𝑅𝑅 (√𝑐𝑐F2+ 4(𝛾𝛾ext±)2
(𝛾𝛾int±)2𝑐𝑐ext2 ) − 2𝜙𝜙ext𝑅𝑅𝑅𝑅𝑐𝑐ext (3.1)
where 𝜙𝜙int, 𝜙𝜙ext, 𝛾𝛾int and 𝛾𝛾ext are internal and external osmotic and activity coeffi- cients, respectively, R the molar gas constant, T the absolute temperature, 𝑐𝑐F is FCD and 𝑐𝑐ext the external salt concentration.
The normal extracellular osmolarity of the cartilage is typically ranging between 350 mOsm and 450 mOsm (Urban et al., 1993). In OA, the extracellular osmolarity can reduce to approximately ~270 mOsm (Bush and Hall, 2005, 2003). In hypotonic challenge, the chondrocyte volumes increase fast, in minutes (Figure 3.1a) (Bush and Hall, 2001a; Guilak et al., 2002; Korhonen et al., 2010a, 2010b; Turunen et al., 2012b). This phenomenon occurs due to passive osmotic uptake of water (Lewis et al., 2011; Wehner et al., 2003). After cell swelling, active ion transporters strive to restore cell volume by transporting ions out of the cell (Lang, 2007). This cell vol- ume recovery is called regulatory RVD (Figure 3.1a) (Hoffmann et al., 2009). Since RVD is an active process, it requires energy and it is assumed to be disturbed in situations where energy is limited. In fact, influence of environmental factors, such as consistency of immersion media or temperature, in experiments of the cartilage osmotic loading have been infrequently investigated.
Osmotic challenge affects also significantly the viscoelastic properties of the chondrocytes; in a hypotonic challenge of 125-150 mOsm, instantaneous and equi- librium elastic moduli decrease significantly by 25-50%, and viscosity of the chon- drocytes is reduced (Guilak, 2000). The changes in the density or the structure of cytoskeleton, e.g. in the microtubules and the microfilaments, and intracellular proteins may induce these changes in the viscoelastic properties of the chondro- cytes (Guilak, 2000; Wang et al., 2015).
Confocal microscopy is commonly used to visualize the chondrocyte volume al- terations in response to changes in the extracellular osmolarity (Bush and Hall, 2001a, 2001b; Errington et al., 1997; Kerrigan et al., 2006; Korhonen et al., 2010a;
Turunen et al., 2012b). Tissue explants and isolated chondrocytes were used in some studies (Bush and Hall, 2001a, 2001b), but there are also studies where chon- drocytes were measured in their native environment (Korhonen et al., 2010a;
Turunen et al., 2012b).
29 where 𝜙𝜙int, 𝜙𝜙ext, 𝛾𝛾int and 𝛾𝛾ext are internal and external osmotic and activity coeffi- cients, respectively, R the molar gas constant, T the absolute temperature, 𝑐𝑐F is FCD and 𝑐𝑐ext the external salt concentration.
The normal extracellular osmolarity of the cartilage is typically ranging between 350 mOsm and 450 mOsm (Urban et al., 1993). In OA, the extracellular osmolarity can reduce to approximately ~270 mOsm (Bush and Hall, 2005, 2003). In hypotonic challenge, the chondrocyte volumes increase fast, in minutes (Figure 3.1a) (Bush and Hall, 2001a; Guilak et al., 2002; Korhonen et al., 2010a, 2010b; Turunen et al., 2012b). This phenomenon occurs due to passive osmotic uptake of water (Lewis et al., 2011; Wehner et al., 2003). After cell swelling, active ion transporters strive to restore cell volume by transporting ions out of the cell (Lang, 2007). This cell vol- ume recovery is called regulatory RVD (Figure 3.1a) (Hoffmann et al., 2009). Since RVD is an active process, it requires energy and it is assumed to be disturbed in situations where energy is limited. In fact, influence of environmental factors, such as consistency of immersion media or temperature, in experiments of the cartilage osmotic loading have been infrequently investigated.
Osmotic challenge affects also significantly the viscoelastic properties of the chondrocytes; in a hypotonic challenge of 125-150 mOsm, instantaneous and equi- librium elastic moduli decrease significantly by 25-50%, and viscosity of the chon- drocytes is reduced (Guilak, 2000). The changes in the density or the structure of cytoskeleton, e.g. in the microtubules and the microfilaments, and intracellular proteins may induce these changes in the viscoelastic properties of the chondro- cytes (Guilak, 2000; Wang et al., 2015).
Confocal microscopy is commonly used to visualize the chondrocyte volume al- terations in response to changes in the extracellular osmolarity (Bush and Hall, 2001a, 2001b; Errington et al., 1997; Kerrigan et al., 2006; Korhonen et al., 2010a;
Turunen et al., 2012b). Tissue explants and isolated chondrocytes were used in some studies (Bush and Hall, 2001a, 2001b), but there are also studies where chon- drocytes were measured in their native environment (Korhonen et al., 2010a;
Turunen et al., 2012b).
Figure 3.1: Schematic presentation of osmotic challenge (a) and static compression (b). In osmotic challenge, the chondrocytes swell fast, 1) to 2), and afterwards cell volume strive to recover, 3). Thus, the radius (r) of the chondrocyte first increases and afterwards recovers (a). Static compression changes the shape of chondrocytes. Thus, the initial vertical radius (r1) of the chondrocyte becomes typically smaller (r2), and the horizontal radius becomes typically larger (r3) under static compression (b).
r r
r
1) 2) 3)
a) Osmotic challenge
swelling
b) Static compression
1)
r1
r2 r3 F 2)
stretch
3.2 CHONDROCYTE RESPONSES TO MECHANICAL LOAD
It has been known for a long time that compressive mechanical loading of the artic- ular cartilage changes the shape of the chondrocytes, the intercellular spacing (Fig- ure 3.1b) (Broom and Myers, 1980), and the chondrocyte volume (Clark et al., 2003;
Guilak et al., 1995; Guilak, 2000). The chondrocytes are sensitive to the mechanical load; dynamic loading with high frequency stimulates the ECM synthesis, while static loading reduces it (Bachrach et al., 1995; Jones et al., 1982; Palmoski and Brandt, 1984). Thus, the mechanical loading significantly regulates chondrocyte metabolic activity (Urban, 1994). Therefore, the composition and structure of the ECM reflect the responses of the chondrocytes to the magnitude and the frequency of loading.
Confocal microscopy is often used for mechanical loading measurements of the articular cartilage and live chondrocytes (Guilak et al., 1995; Han et al., 2009, 2010).
Additionally, light microscopy or stereological tools for histologically fixed com- pressed articular cartilage have been used (Buschmann et al., 1996; Clark et al., 2003). Isolated cells, cartilage explants and fully intact osteochondral samples have been used in mechanical loading measurements of articular cartilage (Broom and Myers, 1980; Buschmann et al., 1996; Clark et al., 2003; Freeman et al., 1994; Han et al., 2009, 2010; Lee et al., 2000). A micropipette aspiration method has been applied to determine mechanical properties of the isolated chondrocytes (Jones et al., 1999).
3.3 CELL – TISSUE INTERACTIONS
Mechanical, environmental and genetic signals regulate the metabolic activity of the chondrocytes (Guilak, 2000). The PCM is a narrow region in the articular cartilage, which consists of collagens, mainly type VI, but also types II and IX, and PGs such as aggrecan, hyaluronan, decorin and fibronectin, around the chondrocytes (Poole, 1997). Thus, the PCM modulates the micromechanical environment of the chondro- cytes (Alexopoulos et al., 2005; Korhonen and Herzog, 2008; Wilusz et al., 2014). It can also transduce the biochemical and biomechanical signals for the chondrocytes (Guilak et al., 2006).
The results of computational models (Julkunen et al., 2009; Korhonen and Her- zog, 2008; Sibole and Erdemir, 2012) and experimental studies (Bush and Hall, 2001a; Choi et al., 2007; Knight et al., 1998) indicate significant roles of the PCM and ECM in controlling the mechanical and physiochemical environments of the chon- drocytes, having in turn an affect on the articular cartilage homeostasis via chon- drocyte metabolism. Several studies have indicated specific roles of the PGs and the collagens of the PCM and ECM to control cell responses (Buschmann et al., 1995, 1996; Lee et al., 2002; Urban et al., 1993; Urban and Bayliss, 1989). For instance, a reduced PG content and, by implication, a decreased FCD content in the PCM of
31
3.2 CHONDROCYTE RESPONSES TO MECHANICAL LOAD
It has been known for a long time that compressive mechanical loading of the artic- ular cartilage changes the shape of the chondrocytes, the intercellular spacing (Fig- ure 3.1b) (Broom and Myers, 1980), and the chondrocyte volume (Clark et al., 2003;
Guilak et al., 1995; Guilak, 2000). The chondrocytes are sensitive to the mechanical load; dynamic loading with high frequency stimulates the ECM synthesis, while static loading reduces it (Bachrach et al., 1995; Jones et al., 1982; Palmoski and Brandt, 1984). Thus, the mechanical loading significantly regulates chondrocyte metabolic activity (Urban, 1994). Therefore, the composition and structure of the ECM reflect the responses of the chondrocytes to the magnitude and the frequency of loading.
Confocal microscopy is often used for mechanical loading measurements of the articular cartilage and live chondrocytes (Guilak et al., 1995; Han et al., 2009, 2010).
Additionally, light microscopy or stereological tools for histologically fixed com- pressed articular cartilage have been used (Buschmann et al., 1996; Clark et al., 2003). Isolated cells, cartilage explants and fully intact osteochondral samples have been used in mechanical loading measurements of articular cartilage (Broom and Myers, 1980; Buschmann et al., 1996; Clark et al., 2003; Freeman et al., 1994; Han et al., 2009, 2010; Lee et al., 2000). A micropipette aspiration method has been applied to determine mechanical properties of the isolated chondrocytes (Jones et al., 1999).
3.3 CELL – TISSUE INTERACTIONS
Mechanical, environmental and genetic signals regulate the metabolic activity of the chondrocytes (Guilak, 2000). The PCM is a narrow region in the articular cartilage, which consists of collagens, mainly type VI, but also types II and IX, and PGs such as aggrecan, hyaluronan, decorin and fibronectin, around the chondrocytes (Poole, 1997). Thus, the PCM modulates the micromechanical environment of the chondro- cytes (Alexopoulos et al., 2005; Korhonen and Herzog, 2008; Wilusz et al., 2014). It can also transduce the biochemical and biomechanical signals for the chondrocytes (Guilak et al., 2006).
The results of computational models (Julkunen et al., 2009; Korhonen and Her- zog, 2008; Sibole and Erdemir, 2012) and experimental studies (Bush and Hall, 2001a; Choi et al., 2007; Knight et al., 1998) indicate significant roles of the PCM and ECM in controlling the mechanical and physiochemical environments of the chon- drocytes, having in turn an affect on the articular cartilage homeostasis via chon- drocyte metabolism. Several studies have indicated specific roles of the PGs and the collagens of the PCM and ECM to control cell responses (Buschmann et al., 1995, 1996; Lee et al., 2002; Urban et al., 1993; Urban and Bayliss, 1989). For instance, a reduced PG content and, by implication, a decreased FCD content in the PCM of
