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PROGRESS IN HISTOCHEMISTRY AND CYTOCHEMISTRY

Progress in Histochemistry and Cytochemistry 45 (2011) 239293
www.elsevier.de/proghi
Review
Impact of sex hormones, insulin, growth factors and peptides on cartilage health and disease
Horst Claassen a,,1 , Martin Schicht a,1 , Friedrich Paulsen a,b,2
a Institut fr Anatomie and Zellbiologie, Martin-Luther-Universitt Halle-Wittenberg, Groe Steinstrae 52, D-06097 Halle (Saale), Germany b Anatomisches Institut II, Friedrich-Alexander-Universitt Erlangen-Nrnberg, Universittsstrae 19, D-91054 Erlangen, Germany
Accepted 21 October 2010

Dedicated to Professor Dr. med. Dr. h.c. Johannes W. Rohen on the occasion of his 90th birthday (September, 18, 2011).
Abstract
Sex hormones contribute to the pathogenesis of osteoarthritis (OA) in both sexes. OA is normally not seen in pre-menopausal women, whereas men may develop the disease as early as the 30th year of life. OA also shows increased incidence in association with diseases such as diabetes mellitus. Recent years have seen characterization of essential components of a functional endocrinal network in the articular cartilage comprising not only sex hormones but apparently insulin, growth factors and various peptides as well. In this review, we summarize the latest information regarding the inuence of sex hormones, insulin, growth factors and some peptides on healthy cartilage and their involvement in osteoarthritis. Both animal and human research data were considered. The results are presented in
Corresponding author. Department of Anatomy and Cell Biology, Martin Luther University Halle-Wittenberg, Groe Steinstr. 52, 06097 Halle (Saale), Germany. Tel.: +00493455571708; fax: +00493455571700. E-mail address: horst.claassen@medizin.uni-halle.de (H. Claassen). 1 Department of Anatomy and Cell Biology, Martin Luther University Halle-Wittenberg, Grosse Steinstr. 52, 06097 Halle (Saale), Germany. 2 Department of Anatomy II, Friedrich-Alexander-Universitt Erlangen-Nrnberg, Universittsstr. 19, 91052 Erlangen, Germany.
0079-6336/$ see front matter 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.proghi.2010.11.002
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H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293
an information matrix that identies what is known, with supporting references, and identies areas for further investigation. 2010 Elsevier GmbH. All rights reserved. Keywords: Cartilage; Articular cartilage; Fibrous cartilage; Hyaline laryngeal cartilage; Sex hormones; Sex hormone receptors; Estrogen; 17-estradiol; Estrogen receptor antagonist; Artifcially induced sex hormone deciency; Estrogen replacement therapy; Ovariectomy; Castration; Testosterone; Androgens; Androgen deciency; Progesteron; Pregnancy; Insulin; Osteoarthritis; Cytokines; Interleukin-1; Tumor necrosis factor alpha; VEGF; Antimicrobial peptides; Trefoil factor family peptides; TFF3; Insulin-like growth factor-I; Transforming growth factor-; Cartilage degrading enzymes; Diabetes mellitus
Contents
1. 2. 3. 4. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Articular cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Osteoarthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sex hormones in cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Detection of sex hormone metabolism enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Sex hormone receptors and the inuence of sex hormones . . . . . . . . . . . . . . . . . . . . 4.2.1. Fibrous cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Hyaline laryngeal cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. Growth joints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.4. Articular cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sex hormones and osteoarthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Negative inuence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Inuence of estrogen receptor antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Positive inuence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Summary: inuence of 17-estradiol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Progesterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Testosterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7. Inuence of an articially induced sex hormone deciency . . . . . . . . . . . . . . . . . . . 5.7.1. Ovariectomy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7.2. Summary: inuence of articially induced estrogen deciency . . . . . . . . 5.7.3. Castration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7.4. Summary: inuence of articially induced androgen deciency . . . . . . . 5.8. Clinical studies on the inuence of estrogen replacement therapy or estrogen/progesterone combination therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8.1. No inuence or negative inuence of estrogen replacement therapy . . . 5.8.2. Positive inuence of estrogen replacement therapy . . . . . . . . . . . . . . . . . . 5.8.3. Summary: inuence of estrogen replacement therapy . . . . . . . . . . . . . . . . 5.8.4. Estrogen/progesterone combination therapy . . . . . . . . . . . . . . . . . . . . . . . . . 5.9. Clinical studies on the inuence of testosterone on articular cartilage . . . . . . . . . . Sex hormones and cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Cartilage-degrading (catabolic) cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 243 243 244 244 244 245 245 246 246 247 254 260 262 262 263 265 265 265 265 267 271 271 271 271 271 273 273 273 273 276
5.
6.
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7. 8.
9.
6.1.1. Interleukin-1beta (IL-1) and tumor necrosis factor-alpha (TNF-) . . . 6.1.2. Vascular endothelial growth factor (VEGF) . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.3. Antimicrobial peptides (AMP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.4. Trefoil factor family peptide 3 (TFF 3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Cartilage-synthesizing (anabolic) cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.1. Insulin-like growth factor-I (IGF-I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.2. Transforming growth factor-beta (TGF-) . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Summary: inuence of the interaction of sex hormones and cytokines . . . . . . . . . Cartilage-degrading enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Summary: inuence of cartilage-degrading enzymes . . . . . . . . . . . . . . . . . . . . . . . . . Estrogens and insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Inuence of insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Osteoarthritis and diabetes mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. Summary: inuence of insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
276 277 277 278 278 278 279 279 279 280 280 280 282 282 282 283 283
1. Introduction
The reaction of cartilage to administration of estrogens has been known for some time (Zondek, 1936). Despite identication in the meantime of sex hormone receptors for 17-estradiol, progesterone and testosterone on human chondrocytes (Ben-Hur et al., 1997) and immortalized human and chondrocytes (chondrocyte cell lines C-28/I2 and T/C-28a2) (Claassen et al., 2010b), little is known to date regarding the metabolic pathways by which the sex hormones inuence cartilage metabolism. In particular, the role of 17-estradiol in postmenopausal osteoarthritis remains unexplained. The role played by insulin in cartilage metabolism has also received little investigative attention. This is astonishing in view of the fact that the extracellular matrix of cartilage is made of sugars and that medical science has been aware for many years now of a relatively high incidence of osteoarthritis in diabetics (Bora and Miller, 1987). A large body of experimental studies have shown that sex hormones, and estrogens in particular, do not unfold their effects in solitary action, but rather in association with serum factors, especially cytokines. 17-estradiol and testosterone interact with interleukin (IL)-1 to induce IL-6, a cytokine with a regulatory function, in inammatory processes in osteoarthritic cartilage (Guerne et al., 1990). For this article, we were given the task of reviewing our own contributions to this area. We have also suggested further avenues of investigation that will be necessary to gain a better understanding of the biology and pathobiology of sex hormones, insulin and cytokines on cartilage, the objective being to delineate approaches to future therapeutic strategies for postmenopausal osteoarthritis.
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2. Cartilage
Cartilage tissue is comprised of specic cells, the chondrocytes, plus the extracellular matrix secreted by these cells. The different types of cartilage include elastic cartilage (e.g. in the external ear), brous cartilage (e.g. in knee joint menisci) and hyaline cartilage (e.g. in the laryngeal cartilages). Articular cartilage can be considered a special form of hyaline cartilage. 2.1. Articular cartilage The cells account for only 1 to 10% of the articular cartilage of adults. The rest is extracellular matrix and interstitial uid. The main components of the extracellular matrix are type II collagen brils, a marker protein of hyaline articular cartilage (Sandell et al., 1992), and proteoglycans (e.g. aggrecan). Type II collagen brils are associated with type IX and XI collagen brils (Eyre et al., 1992; Sandell et al., 2007). Non-calcied articular cartilage features a tangential brous layer adjacent to the articular surface, and beneath this transition and radial ber zones (Fig. 1); it is afxed to the subchondral bone by a thin layer of calcied cartilage (Hunziker, 1992; Sandell et al., 2007). The borderline between non-calcied and calcied articular cartilage is called the tidemark (Hunziker, 1992). Cartilage contains the highest concentration of proteoglycans of all tissue types. Cartilage has solid consistency, can be deformed by pressure to some degree and recovers its original form when the pressure is removed. This biomechanical property, known as elasticity of compression, results from the interaction of proteoglycans and collagen brils (Sandell et al., 2007). Proteoglycans, for example the aggrecan typical of articular cartilage, are high molecular molecules; they are made up of a core protein to which are coupled monomeric sulfated glycosaminoglycans (chondroitin-4 and 6-sulfate, keratin sulfate) with a high water binding capacity. Aggrecan molecules are bound in the extracellular matrix to hyaluronan, a non-sulfated glycosaminoglycan. Hyaluronan is in turn bound to special receptors on the plasma membrane of the chondrocytes, thus anchoring the entire aggrecan molecule to the cell (Heinegard and Pimentel, 1992; Puhl, 1995). Collagen brils are composed of protein chains, which in the typical cartilage collagen type II are wound about one another in a kind of threefold helix (Sandell et al., 2007). The arcade-like arrangement of the type II collagen brils is decisive for the function of this highly specialized tissue. The brils are initially drawn at right angles from the subchondral
Fig. 1. Histology of articular cartilage. The non-mineralized part of the hyaline articular cartilage are divided into a tangential ber zone, a transition zone and a radial ber zone. The tidemark shows the transition from non-mineralized cartilage to the mineralized cartilage zone. The mineralized cartilage zone, which already shows properties of bony tissue, ensures the adhesion articular cartilage to the subchondral bone. The collagen brils are arranged in the typical arcade scheme described by Benninghoff. There are interesting differences between the chondrocytes of the different articular cartilage layers. For instance, the cells of the deep radial ber zone show the highest level of activity in terms of collagen II synthesis (Aydelotte et al., 1992).
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bone in the direction of the cartilage surface, whereby they transit the calcied cartilage and radial ber zones. They then continue on a curved course through the transition zone until they reach the surface of the articular cartilage (tangential ber zone), whereupon they head back down to the bone. The proteoglycans are included in these collagen loops (collagen arcades acc. to Benninghoff) (Fig. 1). Adult articular cartilage has neither innervation nor vascularization. The tissue is nurtured through the synovial by means of diffusion and convection only. Since articular cartilage chondrocytes are no longer capable of high levels of mitotic activity, articular cartilage has a poor regenerative potential.
3. Osteoarthritis
In a healthy state, articular cartilage facilitates a smoothly gliding contact between adjacent joint elements. The term arthrosis designates non-inammatory degenerative joint diseases with inammatory phases in the further course (Puhl, 1995). The patient notices a restriction of movement associated with pain in the affected joints. The primary phenomenon involved in arthrosis is cartilage degradation (loss of hyaline articular cartilage) and cartilage destruction (Walton, 1977). Besides the joint capsule, the subchondral bone is eventually involved as well, for which reason the disease is more accurately termed osteoarthritis (OA) (Goldring and Goldring, 2007). A further sign of involvement of the subchondral bone is the formation of osteophytes. Osteophytes increase the stability of the diseased joint. The extracellular matrix of osteophytes from affected joint segments is characterized by uniform distribution of glycosaminoglycans. By contrast, glycosaminoglycans, in particular the highly sulfated ones, are detected relatively rarely in the matrix of osteophytes in unaffected joint segments (Claassen and Tschirner, 2003; Fig. 2). The signicance of degenerative diseases of the locomotor apparatus in terms of socioeconomics and healthcare policy is increasing parallel to life expectancy. Arthrosis leads to sick leave and attendant damage to national economies. Just how important an intact locomotor apparatus is becomes clear when one considers that the crew of the international space station trains for 2 hours a day to prevent degeneration of connective and supportive tissues in zero gravity. The incidence of OA in the U.S. is around 15% (Simmons et al., 1999). Nearly 27 million U.S. Americans are affected (Stevens-Lapsley and Kohrt, 2010). In Germany, radiological signs of OA (narrowing of the intraarticular space) in 25% of persons over 50 years of age and in over 80% of persons over 75 years of age (Puhl, 1995). A current assessment puts the corresponding count at 5 millions persons in Germany (Vetter, 2010). Crepitation (creaking) of the joint during movement and radiologically veried narrowing of the intraarticular space are symptoms of an advanced stage of arthrosis.
4. Sex hormones in cartilage

4.1. Detection of sex hormone metabolism enzymes Key sex hormone biosynthesis enzymes have been found in chondrocytes of different species. Pre-puberty growth joint cartilage and articular cartilage in rabbits can convert
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Fig. 2. Osteophytes from loaded and unloaded zones of articular cartilage. Osteophytes very often arise at the weight bearing lateral and medial femoral head (Claassen and Tschirner, 2003). Osteophytes from loaded, weight bearing zones of joint cartilage contain more blue-stained glycosaminoglycans (a) than osteophytes from unloaded, non-weight bearing zones (b). This can be considered an example of the principle of function adaptation of the articular cartilage: More glycosaminoglycans are produced in loaded zones. Glycosaminoglyans, as components of the macromolecular proteoglycan molecule, ensure the elastic properties of articular cartilage tissue because of their water storage capacity. Toluidine blue staining (a), Azan staining (b). Magnication a, b x6.
testosterone into dihydrotestosterone, 4 -androstenedione and androstenediol (Takahashi et al., 1984). It can therefore be concluded that both of these cartilage possess the enzymes needed to metabolize testosterone. The chondrocytes of female rabbits also express 17hydroxysteroid dehydrogenase, an enzyme that converts estrone into estradiol (Bellino, 1992). Cartilage cells from the rib growth joint or mandibular joint of male and female rats express the aromatase at the mRNA and protein level required to produce estradiol (Sylvia et al., 2002; Yu et al., 2006). Endogenous estrogen synthesis has been conrmed at mandibular joint chondrocytes (Yu et al., 2009a) and at human cartilage cell line HCS-2/8 chondrocytes (Chagin et al., 2006). 4.2. Sex hormone receptors and the inuence of sex hormones 4.2.1. Fibrous cartilage A recently published study by Wang et al. (2009) revealed clear differences in the distribution of the sex hormone receptors estrogen (ER)-alpha, ER-beta, the relaxin receptors LGR7 (leucin-rich G-protein-coupled receptor 7) and LGR8 as well as progesterone (PR) depending on the localization of the brous cartilage. Fibrous cartilage from the mandibular joint disc showed raised levels: 2.8-fold higher for ER-alpha, 3-fold higher for ER-beta, 3-fold higher for LGR7 and 1.8-fold higher for the PR expression level than in brous cartilage of the knee joint meniscus. Only the LGR8 expression levels in the mandibular joint cartilage were lower than in meniscus cartilage by a factor of 0.5. The expression prole in the mandibular joint disc was comparable to brous cartilage from the symphysis. The ndings were also conrmed by the authors at the protein level. Gender differences were detectable for ER isoforms but not for LGR7, LGR8 and PR. In a further study, the working groups demonstrated that ER-beta, the 6-kDa polypeptide hormone relaxin and
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a combination of ER-beta and relaxin result in a signicant loss of glycosaminoglycans and collagens in the symphysis and in the mandibular joint disc, but not in the knee joint meniscus, when the hormones were administered subcutaneously with a pump (Hashem et al., 2006). The potentiating effect of relaxin appears to be based on an induction of the matrix metalloproteinases (MMP) 1, 3, 9 and 13, which initiate loss of glycosaminoglycans by means of collagen degradation (Kapila et al., 2009). In non-pregnant rats, Samuel et al. (1996) showed, on the other hand, that relaxin exerts an effect on the amount of collagen in the pubic symphysis, which effect is increased by estrogen and eliminated by progesterone. In the paper by Samuel et al. (1996), the question of whether these ndings are signicantly related to the loosening of the symphysis during birth remains hypothetical. 4.2.2. Hyaline laryngeal cartilage Due to the extremely slow ossication of the bony laryngeal skeleton, it makes a good object for study of the effect of sex hormones. The fact that only men develop a pomus adami led to the assumption of a special signicance of sex hormones in ossication of the thyroid cartilage (Pschel and Nowakowski, 1954). However, a number of studies found no evidence of estrogen receptors on the laryngeal cartilages (Reiner et al., 1988; Ferguson et al., 1987; Claassen, 1999; Claassen et al., 2006b), while others did nd estrogen receptors there (Holt et al., 1986; Sheridan et al., 1985). Results for detection of androgen receptors were also sometimes negative (Reiner et al., 1988) and sometimes positive (Tuohimaa et al., 1981; Claassen et al., 2006b). The signicance of androgens (Pschel and Nowakowski, 1954; Beckford et al., 1985; Sassoon et al., 1986; Claassen et al., 2006b) and estrogens (Takahashi and Noumura, 1987) in cartilage growth and the further development of the laryngeal skeleton has not been generally recognized. 4.2.3. Growth joints Sex hormone receptors have been found on cartilage cells from growth plates in humans (Ben-Hur et al., 1993, 1997), as well as in mice (Pinus et al., 1993) and rats (Pinus et al., 1993; Nasatzky et al., 1994). mRNA of the estrogen receptors and is expressed in the primordial cartilage of the ribs and vertebrae of mouse embryos (Lemmen et al., 1999). The estrogen receptor was detected immunohistochemically in the hypertrophic zone, but not in the resting and proliferation zones of the growth joints in young women (Nilsson et al., 1999). 4.2.3.1. Inuence of 17-estradiol. Lebovitz and Eisenbarth (1975), as well as Silbermann (1983), summarized early results on the inuence of sex hormones on growth joint cartilage. Both authors emphasized that on way in which estrogens exert their inuence on growth joints is by inhibiting somatomedin synthesis. Regarding the effect of sex hormones on the chondrocytes of growth joints, it is known that the growth joints show earlier closure under the inuence of estrogens in women as compared to men. Under the inuence of the more pronounced stimulation of alkaline phosphatase compared to men, 17-estradiol results in more rapid maturation of growth joint cells in women (Nasatzky et al., 1993). In ovariectomized rats, estrogen treatment causes an increase in thickness of the growth joint (Turner et al., 1994). The incorporation of radiolabeled sulfate in rib cartilage cells in mice is inhibited in culture medium in the presence of estrogens (Herbai, 1971).
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Chondrocytes from the costochondral growth zone in female rats show a higher binding capacity for 17-estradiol than those from male rats (Nasatzky et al., 1994). This hormone is thought to reduce the membrane uidity of costochondral growth joint cells in female rats by way of a non-genomic effect (Schwartz et al., 1996). This membrane effect of 17estradiol is said to be mediated via an activation of protein kinase C by phospholipase C and G proteins (Silvia et al., 2000). In male rats, 17-estradiol inhibits cell proliferation of chondrocytes in growth joints and thus suppresses longitudinal growth (Cikos et al., 1994). 4.2.3.2. Inuence of testosterone. The growth joint cartilage of hypophysectomized rats shows a metabolic shift in the direction of early aging under the inuence of administration of testosterone (Fahmi et al., 1971; Tarsoly, 1976). Testosterone raises 3 H-thymidine uptake as a proliferation marker in metacarpal growth plates of male sheep (Peralta et al., 1994). Testosterone raises mitotic rates before supporting maturation in growth joint cartilage of male rats (Fahmy et al., 1971). More recent studies have suggested that testosterone reduces 3 H-thymidine uptake and enhances differentiation by stimulating alkaline phosphatase in costochondral growth zones in male rats (Schwartz et al., 1994). Testosterone and dihydrotestosterone cause a signicant increase in DNA synthesis in growth joint chondrocytes of male fetuses (Carrascosa et al., 1990). The also show age-dependent stimulation of uptake of [35 S]-sulfate as an expression of proteoglycan synthesis in cultured growth joint chondrocytes from boys and girls (Blanchard et al., 1991). 4.2.4. Articular cartilage Sex hormone receptors have also been discovered on the articular cartilage cells of various other species (pig, cattle, human) using immunohistochemical methods (Claassen et al., 2001) (Fig. 3). Interestingly enough, both ER-positive and ER-negative chondrocytes occur in all articular cartilage layers. Cultured primary human articular cartilage cells express and estrogen receptors as well as the androgen receptor at the mRNA and protein level (Claassen et al., 2010b). The estrogen receptor was detected on cultured rabbit articular cartilage cells (Rosner et al., 1982a), which receptor shows a higher afnity to its ligand during puberty than before puberty (Dayani et al., 1988). Chondrocytes of the caput mandibulae of female baboons (Sheridan et al., 1985; Aufdemorte et al., 1986) and rats of both sexes (Ng et al., 1999; Yu et al., 2009b) possess estrogen receptors and . Male rats show, astoundingly enough, a higher level of expression of both estrogen receptors than the female animals (Yu et al., 2009). In female baboons, the articular disc of the mandibular joint also contains estrogen receptors (Aufdemorte et al., 1986). Estrogen receptors, but no androgen or progesterone receptors, were found on knee joint chondrocytes of adult dogs (mongrel strain) (Young and Stack, 1982). In contrast to the results obtained for chondrocytes from the cartilaginous laryngeal skeleton, growth joints and proliferation zones, which seem to point in one direction, the inuence of sex hormones on articular cartilage remains unclear and many of the results in this area are contradictory. 4.2.4.1. 17-estradiol. 4.2.4.1.1. No inuence or negative inuence. Mackintosh and Mason (1988), who investigated the articular cartilage of cattle and found no estrogen receptors, reported that
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Fig. 3. Immunohistochemical detection of the estrogen receptor (ER) in articular cartilage tissue of very elderly human subject. ER-positive and ER-negative chondrocytes are detected in both the surface (a) and deep (b) articular cartilage layers (arrows) (Claassen et al., 2001). a: 75-year-old man, b: 83-year-old woman. Magnication a, b: bar 27,5 m.
estrogens had no inuence on the metabolism of articular cartilage cell. No relationship was found between the reduction of tibial cartilage volume and an estrogen replacement therapy in healthy postmenopausal women (Wluka et al., 2004). Cultured rabbit joint cartilage cells react to addition of TGF-1, but not to addition of 17-estradiol, with increased synthesis of collagen II and glycosaminoglycans (Ab-Rahim et al., 2008). Combined administration of TGF-1 and 17-estradiol in no way alters the positive effect of TGF-1. 6-month-old ER/ knockout mice show an increase in osteophytes, but the articular cartilage damage does not differ from what is observed in wild type animals (Sniekers et al., 2009). Priest and Koplitz (1962) found a negative inuence on articular cartilage metabolism in the form of suppression of proteoglycan synthesis or reduction of uptake of radiolabeled sulfur in rats, as did Rosner et al. (1979) in rabbits. 17-estradiol reportedly showed a cytotoxic effect on cultured rabbit articular cartilage cells, which effect was mitigated by application of the enzyme catalase (Tsai et al., 1998). In cultured rabbit chondrocytes, higher doses of estradiol (105 M) inhibit DNA synthesis (Corvol et al., 1972). Higher doses of estradiol (108 M-106 M) result in a reduction of cartilage thickness on the caput mandibulae, as well as in a reduction of the proteoglycan content, in female rats (Ng et al., 1999). High doses of estradiol suppress uptake of 3 H-thymidine in cultured
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Fig. 4. Generation of reactive oxygen species (ROS) in cultured bovine articular chondrocytes. ROS were generated with Fe2 SO4 , vitamin C, and hydrogen peroxide dissolved in HBSS. The nal concentration of Fe2 SO4 was 3 M, whereas those of vitamin C and of hydrogen peroxide were 100 g/ml and 880 M, respectively. The release of thiobarbituric-acid-reactive substances (TBARS, lipid peroxidation) and lactate dehydrogenase (LDH, membrane damage) was measured photometrically. In chondrocytes incubated repeatedly with 17-estradiol, the production of TBARS, and LDH release, were signicantly suppressed. Estradiol was assumed to accumulate in the plasma membrane and to decrease membrane uidity resulting in protection against lipid peroxidation (Claassen et al., 2005).
human chondrocytes (Scranton et al., 1975). Estrogens are reportedly chondrodestructive in adipose women (Tsai and Liu, 1992). 4.2.4.1.2. Positive inuence. Several working groups describe a positive inuence of estrogens on cartilage. An age-dependent positive inuence on the uptake of radiolabeled sulfur in proteoglycans is demonstrated in rabbit articular cartilage (Corvol et al., 1987). In rabbit rib cartilage chondrocytes, preincubation of the cells with 17-estradiol further incubation with IGF-I results in increased DNA synthesis (Itagane et al., 1991). Estrogens possess a radical-scavenging property (Claassen et al., 2005). We developed a culturing system for specic production of oxygen radicals with bovine articular cartilage cells (Fig. 4). After several applications of 17-estradiol, the plasma membrane of the chon-
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drocytes stabilizes, thus better protecting the chondrocytes from attack by oxygen radicals. In cultured articular cartilage explants from cattle, 17-estradiol inuences anabolic and catabolic genes, whereby the global effect of the hormone is categorized as positive (Sniekers et al., 2010). 17-estradiol stimulates the uptake of radiolabeled sulfate and enhances the activity of alkaline phosphatase as well as the activity of protein kinase C (PKC) in 2ndpassage articular cartilage chondrocytes from women (Kinney et al., 2004). Chondrocytes from women, but not from men, react to application of 17-estradiol with increased DNA synthesis and increased activity of alkaline phosphatase as well as with increased incorporation of radiolabeled sulfate (Kinney et al., 2005). Integration of cultured bovine articular cartilage blocks is improved by addition of 17-estradiol, which could be of signicance in treatment of traumatic cartilage damage in particular (Englert et al., 2006). Estrogens exert a positive inuence on the regenerative potential of chondrogenic progenitor cells occurring in osteoarthritic cartilage (Koelling and Miosge, 2010). 4.2.4.1.3. Pregnancy. In the animal model pregnant rabbit, important specic molecules for articular cartilage such as type II collagen, biglycan, collagenase and tissue inhibitor of metalloproteinases (TIMP) -1 are reduced at the mRNA level under the inuence of estrogens and relaxin (Hellio le Graverand et al., 1998). 4.2.4.1.4. Summary: inuence of 17-estradiol. Summarizing the results obtained on the inuence of 17-estradiol on articular cartilage chondrocytes does not produce a uniform picture (Table 1). The results depend on the species studied and on the hormone application. Administration of estrogens in rabbits produces a notable preponderance of negative effects on the articular cartilage. A further observation is that 17-estradiol often does not show an effect until it combined with other hormones, for instance insulin or relaxin. Fig. 5a and b summarize important ndings made of other working groups. Figure 5c explains the abbreviations. Fig. 5a and 5b show the inuence of 17-estradiol on various intracellular metabolic pathways. It then becomes clear that 17-estradiol stimulates the uptake of radiolabeled sulfate in women and increases the activity of alkaline phosphatase, and can also induce IL-1. Fig. 6 shows a corresponding scheme with our own research results. It becomes clear that 17-estradiol stabilizes the plasma membrane of articular cartilage cells and, in cultured articular cartilage cells from female patients, suppresses the expression of MMP 3 and 13 at the mRNA level. Surprisingly, the cartilage matrixdegrading enzyme MMP 1 is already detected in the articular cartilage of a 16-year-old man (Claassen et al., 2010a) (Fig. 7). It is thus not surprising that immunohistochemical detection result for this enzyme in the territorial matrix of the articular cartilage of a very elderly woman is also positive. 4.2.4.2. Testosterone. 4.2.4.2.1. No inuence or negative inuence. Testosterone does not inuence 35 Slabelled proteoglycan synthesis in articular cartilage cells of cattle (Mackintosh and Mason, 1988). In male Wistar rats, testosterone reportedly furthers the maturation and degeneration of the articular cartilage while stimulating the alkaline phosphatase (Tarsoly and Mateescu, 1972). No connection between the serum testosterone level and knee joint cartilage volume is found in middle-aged women (Hanna et al., 2007). 4.2.4.2.2. Positive inuence. Several working groups report a positive effect of testosterone on articular cartilage. Chondrocytes from rabbits of both sexes reportedly
Table 1. Experimental studies on the inuence of 17-estradiol on articular cartilage cells. Species pig, cow, human rat Investigative method cell culture cell culture Parameters immunohistochemistry uptake of radiolabeled sulfur, proteoglycan synthesis cartilage thickness, proteoglycan content DNA synthesis uptake of radiolabeled sulfur uptake of radiolabeled sulfur, proteoglycan synthesis DNA synthesis Special features detection of estrogen receptor high 17-estradiol doses: 108 M-106 M high 17-estradiol doses: 105 M Evaluation of inuence of 17-estradiol negative Author
Claassen et al., 2001 Priest and Koplitz 1966
female rat rabbit rabbit rabbit
animal model cell culture cell culture cell culture
negative negative positive negative
Ng et al., 1975 Corvol et al., 1972 Corvol et al., 1972 Rosner et al., 1979
rabbit
cell culture
rabbit female rabbit
cell culture cell culture
rabbit
cell culture
type II collagen, biglycan, collagenase and TIMP-1 at mRNA level DNA synthesis
preincubation with 17-estradiol, further incubation with IGF-I pregnancy: highs 17-estradiol + Relaxin incubation with TGF-1
positive
Itagane et al., 1991
negative negative
Tsai et al., 1998 Hellio le Graverand et al., 1998
17-estradiol: neutral, positive on collagen II synthesis, positive on glycosaminoglycan synthesis
Ab-Rahmin et al., 2008
251
252
Table 1. (Continued ) Species cows female cows Investigative method cell culture cell culture Parameters metabolism detection of radical damage due to production of TBARs and LDH release uptake of radiolabeled proline, type II collagen synthesis due to ELISA uptake of radiolabeled sulfate Special features repeated application of 17-estradiol Evaluation of inuence Author of 17-estradiol
neutral stabilization of plasma membrane
Mackintosh and Mason, 1988 Claassen et al., 2005
female cows
cell culture
preincubation with 17-estradiol, further incubation with Insulin preincubation with 17-estradiol, daidzein or genistein, further incubation with Insulin high 17-estradiol doses second-passage articular cartilage cells
negative
Claassen et al., 2006
female cows
cell culture
17-estradiol: neutral, daidzein: positive, genistein: positive negative negative positive
Claassen et al., 2008
human adipose women women
cell culture clinical trial cell culture
Postmenopausal women women
estrogen replacement therapy cell culture
uptake of radiolabeled thymidine uptake of radiolabeled sulfate, activity alkaline phosphatase and PKC tibial cartilage volume
Scranton et al., 1975 Tsai and Liu 1992 Kinney et al., 2004
neutral
Wluka et al., 2004
DNA synthesis, uptake of radiolabeled sulfate, activity alkaline phosphatase
positive
Kinney et al., 2005
Table 1. (Continued ) Species human women and men Investigative method cell culture cell culture, Real time RT-PCR Parameters DNA synthesis MMP-1, -3, and 13, TIMP-1 and 2 at mRNA level detection of receptor proteins of ER/, AR and IR Special features Evaluation of inuence Author of 17-estradiol Yu et al., 2009a Claassen et al., 2010a
women, immortalized cell lines C-28/I2 and T/C-28a2
cell culture, RT-PCR, Western Blot
positive positive primary human articular cartilage cells, three dimensional alginate culture points of reference for inuencing the signal transduction path of the insulin receptor
Claassen et al., 2010b
253
254
react to testosterone with an age-dependent stimulatory effect on 35 S-labelled proteoglycan synthesis (Corvol et al., 1987, 1997). Again in rabbits, preincubation of rib cartilage chondrocytes with testosterone and further incubation with IGF-I results in increased uptake of radiolabeled sulfate (Itagane et al., 1991). Androgens stimulate collagen II and proteoglycan synthesis in human chondrocytes (Franchimont and Basler, 1991). Application of testosterone to organ cultures of bovine joint cartilage results in improved integration of the cartilage transplant, as evidenced e.g. by an increased glycosaminoglycan content (Englert et al., 2006). 4.2.4.2.3. Summary: inuence of testosterone. The Inuence of testosterone on chondrocytes can be characterized as mainly positive. This is also true for rabbits (Table 2). It is particularly notable that a positive effect in rabbits occurs with the following experimental setup: preincubation of chondrocytes with 17-estradiol, further incubation with IGF-I.
5. Sex hormones and osteoarthritis

A number of factors contribute to the pathogenesis of OA, ranging from incongruous articular surfaces to nutrition to hormonal inuences (Fig. 8). The latest insights suggest that acute damage by injury or infection may initiate joint destruction (Vetter, 2010). Besides aging, gender-specic risk factors are also seen as culpable in the pathogenesis of OA. The incidence curve of the disease spikes upwards in men over 30 and women over 50. The
Fig. 5. a Foreign results (1) concerning the inuence of 17-estradiol and testosterone on intracellular pathways of articular chondrocytes. The results of the respective workgroups are marked with Arabic numerals. 1: In second passage articular cartilage cells from women, 17-estradiol stimulates the uptake of radiolabeled sulfate, increases the activity of alkaline phosphatase and PKC (Kinney et al., 2004). 2: In male Wistar rats, testosterone is said to encourage maturation and degeneration of the articular cartilage under stimulation of the alkaline phosphates (Tarsoly and Mateescu, 1972). 3: 17-estradiol and testosterone function in combination with IL-1 to induce IL-6, a cytokine with a regulatory function in inammatory processes in osteoarthritic cartilage (Guerne et al., 1990). 9: In pregnant rabbits, the inuence of 17-estradiol and relaxin results in reduced expression of type II collagen, collagenase, TIMP-1, TNF- and COX-2 (Hellio le Graverand et al., 1998). Fig. 5b Foreign results (2) concerning the inuence of cytokines on intracellular pathways of articular chondrocytes. The results of the respective workgroups are marked with Arabic numerals. 4: A raised prostaglandin level E(2) in a case of osteoarthritis is accompanied by increased proteoglycan degradation (Hardy et al., 2002). 5: Due to activation of the transcription factor NF-kB, more MMP-3 and 13 are produced under the inuence of IL-1 (Schulze-Tanzil et al., 2004; Ahwad et al., 2007). 6: IL-1 causes a rise in the level of the enzyme cyclooxygenase-2 (COX-2), which is responsible for production of prostaglandins that contribute to the pathogenesis of osteoarthritis. 7: TNF- induces production of the enzyme COX-2 at the mRNA level, which contributes to production of prostaglandins (Geng et al., 1995). 8: VEGF contributes to cartilage-destructive processes within the framework of osteoarthritis since this enzyme increases the production of matrix metalloproteinases (Enomoto et al., 2003) and induces the cartilage-destructive cytokines IL-1 and TNF- (Pufe et al., 2004a). Fig. 5c Receptors and ligands. Here the reader will nd symbols for the receptors, ligands, cytokines, proteins and enzymes used in Fig. 5a and b.
255
256
Fig. 5. (Continued )
257
Fig. 5. (Continued )
258
Fig. 6.
259
latter receive surgical treatment more often than men because of their advanced age (Fig. 9, Claassen et al., 2010a). It appears that women are protected from OA before the menopause. Numerous clinical, pathological and epidemiological studies suggest that women suffer more from OA after menopause than before (Schouten et al., 1992) and that hormones, in particular estrogens and androgens, contribute to disease outbreak (Spector and Campion, 1989; Spector et al., 1991a,b, 1997; Tsai and Liu, 1992c; Wluka et al., 2000; Roman-Blas et al., 2009). The mandibular joint holds a special position in joint disease. 80% of the patients in North America treated for mandibular joint symptoms are women (Wang et al., 2008); a connection with estrogen metabolism is suspected. A study at the University of Pisa with 35 patients of both sexes showed that osteoarthritic changes in the mandibular joint correlate with a changed serum estrogen level, in particular with raised estrogen (Landi et al., 2004, 2005). According to more recent studies, locally synthesized estrogens are said to play a role in the development of an OA (Liu et al., 2009). Occurrence of OA in men climbs starting at the age of 30. Men appear to have no protection against this disease. Earlier studies of mice report a negative effect of testosterone on articular cartilage (Silberberg and Silberberg, 1961, 1963). Recently, a positive correlation between serum testosterone level and cartilage volume of the medial tibial plateau was found in healthy men around 50 years of age (Cicuttini et al., 2003; Hanna et al., 2007). In menopausal women with OA, raised serum testosterone levels were measured in the course of the disease (Makarova and Bobkov, 1999). Johanson (1985) summarized the early results on the signicance of sex hormones in osteoarthritis, but was unable to formulate guidelines for its treatment. In their review,
Fig. 6. Own results of hormones, especially concerning the inuence of 17-estradiol and insulin, or cytokines on intracellular pathways of articular chondrocytes. The results are marked with Roman numerals. I: ER discovered on articular cartilage cells from pig, cow and human (Claassen et al., 2001). II: In a dened culturing system for production of oxygen radicals, 17-estradiol, repeatedly applied, proved to be a radical scavenger similar to vitamin E (Claassen et al., 2005). The hormone probably stabilizes the plasma membrane of the articular cartilage cells. III: In the animal model ovariectomized Gttingen Minipigs, the glycosaminoglycan content in the knee joint is signicantly reduced (Claassen et al., 2002, 2006a). IV: Catabolic cytokines, for example TNF- and IL1-, cause a pronounced induction of human beta-defensin (HBD) 3 in cultured articular cartilage cells (Varoga et al., 2005a/b). In osteoarthritic cartilage, HBD-2, the expression of which is regulated by TNF, IL-1 and IL-6, plays a key role (Varoga et al., 2006). V: In pathologically changed articular cartilage, TFF-3 encourages the degradation of cartilage matrix by up-regulating the matrix metalloproteinase (MMP) 1, 3 and 13, and has a proapoptotic effect (Rsler et al., 2010). VI: In primary human articular cartilage cells from female patients, 17-estradiol in physiological and supraphysiological doses suppresses the expression of MMP-3 and 13 at mRNA level (Claassen et al., 2010a). VII: Insulin increases type II collagen synthesis in cultured bovine articular cartilage cells signicantly (Claassen et al., 2006c). Preincubation of the cells with 17-estradiol results in a signicant suppression of the protein anabolic effect of insulin on proline incorporation and type II collagen synthesis (Claassen et al., 2006c). VIII: Insulin signicantly increases sulfate uptake in cultured bovine articular cartilage cells (Claassen et al., 2008). Preincubation of the chondrocytes with the phytoestrogens daidzein or genistein results in a signicant increase in the anabolic effect of insulin on sulfate uptake (Claassen et al., 2008).
260
Fig. 7. Immunohistochemical detection of matrix metalloproteinase 1 (MMP-1) in articular cartilage of young and aged persons. a: Pericellular matrix of some chondrocytes in the upper and lower radial ber zone showed a positive reaction to antibodies to MMP-1. b: In comparison to a, territorial matrix of several chondrocytes was immunostained by MMP-1 antibodies. Positive staining in articular cartilage from a 16-year-old man (arrows). By contrast, cartilage-degrading enzymes are common in articular cartilage of older people, for instance in the joint tissue of an 87-year-old woman (arrows). Magnication a, b: bar 30 m.
Gokhale et al. (20040) conclude that the studies to date addressing a connection between estrogen and OA show no uniformity. de Klerk et al. (2009) also found no clearly dened relation between serum estradiol levels and OA. Tank et al. (2008), on the other hand, claimed in their review that estrogen therapy may not only be effective in preserving bone tissue, but articular cartilage tissue as well. 5.1. Negative inuence In young mice, strain C57BL6, an injection of 17-estradiol changes the cell organelles in a way that can be observed under the electron microscope: the Golgi apparatus is enlarged (Silberberg et al., 1965). The authors see a correspondence of these changes with defective proteoglycan synthesis. In meniscectomized rabbits, 17-estradiol reportedly exacerbates OA (Rosner et al., 1986). In postmenopausal women, synovial 17-estradiol and a raised binding capacity for this hormone are considered risk factors for OA (Tsai et al., 1992). There is a correlation in women between 17-estradiol and symptoms in the discus articularis of the mandibular joint (Abubaker et al., 1993). A specic genetic constellation of the estrogen
Table 2. Experimental studies on the inuence of testosterone on articular cartilage cells. Species Investigative method Parameters Special features Evaluation of inuence of testosterone negative positive positive Author
male rats female and male rabbit rabbit
animal model cell culture cell culture
alkaline phosphatase, maturation + degeneration proteoglycan synthesis uptake of radiolabeled sulfate proteoglycan synthesis improved uptake of articular cartilage blocks into surrounding tissues synthesis of type II collagen and proteoglycans magnetic resonance imaging, blood analysis
Age-dependence preincubation with testosterone, further incubation with IGF-I -
Tarsoly and Matescu 1972 Corvol et al., 1987, 1997 Itagane et al., 1991
cows cows
cell culture animal model
neutral positive
Mackintosh and Mason, 1988 Englert et al., 2006
human
cell culture
positive
Franchimont and Basler 1991 Hanna et al., 2007
women
clinical observations
positive
261
262
Fig. 8. Pathogenic factors in osteoarthritis (OA). The pathogenesis of OA is divergent with biomechanical and non-biomechanical factors having an effect. The former are characterized, for example, by the deviation of the axis, the incongruence of the articular surfaces, and the properties of the subchondral bone. In addition to heritability and nutrition, hormonal inuences belong to the non-biomechanical factors. Regarding the current view of OA, Professor Dr. med. Wolfgang Rther (Director of the Clinic and Polyclinic for Orthopedic Medicine at the University Clinics, Hamburg-Eppendorf) was quoted in an article in the Deutsches rzteblatt of 29 October 2010 (Vetter, 2010). This statement can serve a supplemental purpose here. Professor Rther said: We always talk about joint wear as the basis of arthrosis. But this does not reect the true facts of the matter. The factors leading up to arthrosis are many and varied: It may be a matter of metabolic dysfunctions, an infection or inammatory processes that damage the cartilage. An arthrosis only develops, he said, when the cartilage has received initial damage, on which basis the joint destruction then proceeds.
receptor, genotype PpXx with the combination PvuI and XbaI, is said to be a signicant risk factor for development of OA (Ushiyama et al., 1998). Greater susceptibility to OA reportedly correlates not only with certain variants of the estrogen receptor, but also with variants of the aromatase gene (Riancho et al., 2010). In organ cultures of chondrocytes from the mandibular joints of mice, 17-estradiol increases the expression of the inammatory cytokines IL-1, IL-6 and IL-8, thus contributing to the pathogenesis of OA (Yun et al., 2008). 5.2. Inuence of estrogen receptor antagonists In the animal model of an arthrosis induced by meniscectomy, the estrogen receptor antagonist tamoxifen reduces articular cartilage damage in male rabbits (Colombo et al., 1983). 5.3. Positive inuence Contrary to the effect of tamoxifen, other working groups report a positive effect of administration of 17-estradiol on articular cartilage, expressed in male mice of the
263
Fig. 9. Age and gender-based distribution of patients with a hip or knee joint endoprosthesis. The diagram refers to the patients who underwent surgery to receive a hip or knee joint endoprosthesis in the year 2000 at the Orthopedic Clinic of Christian Albrecht University of Kiel, Germany. Looking at the spectrum of male and female patients aged 60 to 90 makes it clear that women received a hip or knee joint endoprosthesis much more frequently than men. Of course the specic hormonal conditions to which women are subjected beginning at the age of 50 could explain some of these cases (altered sex hormone metabolism in the menopause). The two patients between 10 and 20 years of age were accident cases.
C57 BLJax6 strain as prevention of development of OA (Silberberg and Silberberg, 1963a/b) and in female mice of the C57 BI6 strain as a reduction of susceptibility to this joint disease (Silberberg and Silberberg, 1970). In female mice of the B10.Q stain that develop arthrosis due to immunization with collagen II, the disease appears early under estrogen receptor blockade with ICI 182.780 (Jansson and Holmdahl, 2001). These results suggest a positive effect of estrogen on arthrosis in this mouse strain. Reduced levels of uCTX-II, a degradation product of type II collagen, was observed under both oral and transdermal estradiol therapy (Ravn et al., 2004). The oxidative stress caused by oxygen radicals that can be produced by any intracellular reaction contributes to the pathogenesis of many degenerative diseases, including OA. In a specic culturing system designed to produce oxygen radicals using Fe2 SO4 , vitamin C and H2 O2 , 17-estradiol proved to be a radical scavenger, similarly to vitamin E (Claassen et al., 2005). The estradiol effect observed here most likely does not occur at the gene level; rather, the accumulating hormone stabilizes the plasma membrane of the chondrocytes (Claassen et al., 2005). 5.4. Summary: inuence of 17-estradiol To summarize, the inuence of 17-estradiol on OA depends on the species under study (Table 3). In mice, administration of estrogens improves the symptoms in most cases, whereas in rabbits the tendency is to worsen the symptoms. Notably, repeated application
Table 3. Experimental studies on the inuence of 17-estradiol on osteoarthritis. Species mice, strain C57BL female mice, strain C57 BI female mice, strain B10.Q Investigative method Parameters animal model animal model animal model, estrogen receptor blockade by ICI 182,780 animal model transmission electron microscopy Special features Evaluation of inuence of 17-estradiol Author
264 H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293
exacerbation, Golgi apparatus enlarged animals susceptible to positive, susceptibility to OA OA reduced arthrosis due to positive, because immunization with exacerbation due to estrogen type II collagen receptor blockade meniscectomized animals meniscectomized animals discus articularis of mandibular joint improvement with use of estrogen receptor antagonist tamoxifen exacerbation
Silberberg et al., 1965 Silberberg and Silberberg, 1970 Jansson and Holmdahl, 2001
male rabbit
histology
Colombo et al., 1983
rabbit
animal model
binding capacity for 17-estradiol analysis of genotype of estrogen receptor UCTX II as degradation product of type II collagen mRNA expression
Rosner et al., 1986
Postmenopausal clinical observations women women clinical observations human human human cows female cows clinical observations clinical observations cell culture animal model cell culture
exacerbation, 17-estradiol is Tsai et al., 1992 risk factor risk factor both 17-estradiol Abubaker et al., 1993 and progesterone risk factor if genotype PpXx Ushiyama et al., 1998 positive, reduction of uCTX II Ravn et al., 2004 blocks cartilage-protective Wang et al., 2010 effect positive, acts on anabolic and Sniekers et al., 2010 catabolic genes stabilization of plasma Claassen et al., 2005 membrane
oral and transdermal estradiol therapy inuence of bisphenol mRNA Expression articular cartilage explants detection of radical damage repeated application due to production of of 17-estradiol TBARs and LDH release
265
of 17-estradiol to female bovine joint chondrocytes stabilizes the plasma membrane. In postmenopausal women the results are not uniform, ranging from identication of estrogen as a risk factors to unfavorable genetic variants of the estrogen receptor and even to positive effects: For instance, 17-estradiol improves levels of the collagen II degradation product CTX-II in the urine. With regard to any therapeutic efcacy of this nding, however, it must be noted that increased levels of CTX-II in the urine do not correlate with progression of OA and occurrence of osteoarthritic damage. 5.5. Progesterone Raised serum progesterone levels (hyperprogesteronemia) were measured in Russian coalminers with OA (Siniachenko et al., 1996). A study at the University of Pisa with 35 patients of both sexes showed that osteoarthritic changes in the mandibular joint do not correlate with changed serum progesterone levels (Landi et al., 2004, 2005). 5.6. Testosterone There also appears to be a connection between androgens and OA (da Silva et al., 1993b). Wilson et al. (1976) reported on a 53-year-old man with a ring-shaped Y-chromosome and hypogonadism who suffered from a severe case of OA requiring bilateral hip joint replacement. Russian coalminers with OA showed reduced serum testosterone levels (Siniachenko et al., 1996). Higher serum testosterone levels in middle-aged men are associated with greater articular cartilage volume, but also with higher levels of cartilage loss later in life (Hanna et al., 2005). 5.7. Inuence of an articially induced sex hormone deciency 5.7.1. Ovariectomy A reduction of proteins in the discus articularis of the mandibular joint is observed in ovariectomized rats following combined administration of estrogen/progesterone, in orchiectomized rats, on the other hand, after sole administration of estrogen (Abubaker et al., 1996). Ovariectomy in young rats results in increased thickness of the articular cartilage in the anterior and central segments of the caput mandibulae of the mandibular joint (Okuda et al., 1996). Findings in ovariectomized rats, in which an increase in the proportion of articular cartilage in the mandibular joint is observed both with and without 17-estradiol substitution, point in the same direction (Yasuoka et al., 2000). On the other hand, studies in rats in which an ovariectomy caused a signicant rise in CTX II as a degradation product of type II collagen, which increase was then mitigated by administering estrogen, point in a different direction (Oestergaard et al., 2006). Bay-Jensen et al. (2009) also report a rise in the collagen II degradation product CTX II in the urine of ovariectomized rats. However, immunohistochemical detection of CTX II in the articular cartilage showed that the estrogen deciency resulted in discrete changes only (Bay-Jensen et al., 2009). It thus remains questionable whether increased urinary CTX II is signicant in terms of the pathogenesis and course of OA. In ovariectomized rats, loss of extracellular matrix is reduced by physiologic doses of estradiol (Ganesan et al., 2008), an estrogen replacement therapy
266
or treatment with estrogen receptor modulators (Gao and Du, 2008). It is further reported that electromagnetic elds result in down-regulation of expression of the enzyme MMP-13, which degrades the articular cartilage matrix in ovariectomized rats (Luo et al., 2008). An articial estrogen deciency caused by ovariectomy results in a breakdown of the articular cartilage in female mice (Da Silva et al., 1998). STR/ort mice are genetically predisposed to develop spontaneous gonarthrosis, whereby the pathological changes are more pronounced in male mice than in female mice (Walton et al., 1977; Mason et al., 2001; Mahr et al., 2003). An ovariectomy, on the other hand, does not result in increased OA in female STR/ort mice (Chambers et al., 2001). Under the experimental conditions of an articially induced estrogen deciency in ovariectomized DBA/1 mice, administration of the estrogen receptor modulator raloxifen results in reduced production of cartilage oligomeric matrix protein (COMP) as a degradation product of the articular cartilage protein type II collagen (Jochems et al., 2007); also reduced under this treatment is the cartilage-destructive cytokine TNF- at the mRNA level. Normal mice with OA induced by meniscus destabilization develop more symptoms of this disease than ovariectomized and meniscus-destabilized mice (Ma et al., 2007). In ovariectomized rabbits with a meniscectomy-induced OA, 17estradiol results in exacerbation and tamoxifen to alleviation of the disease (Rosner et al., 1982b). Tsai and Liu (1992a, b) also report damage to the articular cartilage of ovariectomized rabbits, caused by up-regulation of estrogen receptor following administration of 17-estradiol. Intraarticular injections of 17-estradiol in knee joints of ovariectomized rabbits result in cartilage damage (Tsai et al., 1993). In bilaterally ovariectomized rabbits, the articular cartilage damage is increased, leading to a rise in the Mankin score (Castaneda et al., 2010). It was demonstrated in the animal model ovariectomized and intact Gttingen Minipigs that the relation between low to high sulfated glycosaminoglycans is changed in the extracellular matrix of the articular cartilage post ovariectomy (Claassen et al., 2002). The glycosaminoglycan content of the knee joint cartilage is also signicantly reduced in ovariectomized animals compared to a control group (Claassen et al., 2006a). Reduced glycosaminoglycan content and attendant reduced water storage capacity and loss of elasticity are the potential consequences of estrogen deciency for the articular cartilage. The semiquantitative enzymatic histochemical detection of alkaline phosphatase-positive chondrocytes does not differentiate between articular cartilage samples from ovariectomized Minipigs and control animals (Fig. 10). This nding is probably species-specic, since based on ndings made in cultured articular cartilage cells from women (Kinney et al., 2005), a deciency of alkaline phosphatase, a mineralization metabolism enzyme, would have been expected in estrogen deciency. An ovariectomy is also damaging to the articular cartilage of sheep (Turner et al., 1997). Signicantly fewer severe osteoarthritic lesions occurred in ovariectomized cynomolgus monkeys under estrogen replacement therapy (Ham et al., 2002). In cultured chondrocytes from ovariectomized monkeys, incubation with 17estradiol raises production of insulin-like growth factor binding protein-2 (IGFBP-2) as well as proteoglycan synthesis (Richmond et al., 2000). Dihydrotestosterone slows loss of extracellular articular cartilage matrix in orchiectomized rats (Ganesan et al., 2008). This effect may be mediated by inhibition of aggrecanases (ADAMTS), which belong to the group of metalloproteinases (Wu et al., 2006; Huang and Wu, 2010). Raised synovial levels of insulin-like growth factor-I (IGF-I) and II (IGF-II) as well as insulin-like growth factor binding protein-1 (IGFBP-1) and 3 (IGFBP-3) were observed in ovariectomized cynomol-
267
Fig. 10. Enzymatic histochemical presentation of alkaline phosphatase (AP) in the articular cartilage of intact and ovariectomized Gttingen Minipigs. AP-positive chondrocytes were detected in the radial ber zone near the tidemark and in the zone of mineralized cartilage, but not in the tangential ber zone or in the transition zone (Claassen et al., 2006). Compared with the control (a), no more AP-positive chondrocytes were counted in the group of ovariectomized animals (b). During their formation, chondrocytes that form the zone of mineralized cartilage have properties similar to growth plate chondrocytes. In adults, the mineralized cartilage zone becomes quiescent, but not inactive. This zone may be reactivated in OA and may progressively calcify the unmineralized cartilage under the inuence of alkaline phosphatase as the key enzyme of mineralization processes. Magnication x 40 (a, b).
gus monkeys under estrogen administration, which could also have a positive effect on articular cartilage (Fernihough et al., 1999). Sniekers et al. (2008) investigated animal models for the effects of estrogen deciency induced by ovariectomy. Taking into account only studies with sexually mature animals, 11 of 14 revealed damaging effects of ovariectomy on articular cartilage. 5.7.2. Summary: inuence of articially induced estrogen deciency We have reviewed 24 studies of articially induced estrogen deciency due to ovariectomy (Table 4). The estrogen deciency damaged the articular cartilage in 8 studies, but caused no damage to it in 5 studies. The results were neutral in 11 studies. Regarding estrogen replacement in the ovariectomized animals, an improvement in articular cartilage status was observed in 7 studies, with exacerbation observed in 3 studies. In 14 studies, estrogen therapy in OVX animals showed neutral results. The conclusion can thus be proffered, albeit very cautiously, that ovariectomy-induced estrogen deciency damages articular cartilage and that an improvement is seen under estrogen replacement. The results for ovariectomized rabbits do not correlate well with the others. In rabbits, intraarticular administration of 17-estradiol causes cartilage damage. Systemic administration of 17-estradiol also results in a worsening of the condition of articular cartilage, whereby tamoxifen mitigates the symptoms. It must also be remarked that ovariectomyinduced estrogen deciency is not necessarily the best means of simulating menopausal estrogen deciency. This experimental approach also fails to consider the fact that the adrenal gland continues to synthesize the basic building-blocks of estrogen and testosterone metabolism and to release them into the bloodstream.
268
Table 4. Animal models with an articial estrogen deciency due to ovariectomy (OVX). Species Hormone replacement Result Estrogen deciency harmful? Improvement with estrogen replacement? no Improvement with estrogen receptor modulator? Author
rat (OVX)
estrogen/progesterone
young rat (OVX)
rat (OVX)
rat (OVX)
estrogen
rat (OVX)
rat (OVX)
estrogen
rat (OVX)
physiological 17-estradiol therapy estrogen replacement therapy and treatment with estrogen receptor modulators
rat (OVX)
reduction of proteins in discus articularis of mandibular joint increased articular cartilage thickness in caput mandibulae of mandibular joint increased articular cartilage share in mandibular joint increased articular cartilage share in mandibular joint rise in CTX II as degradation product of type II collagen drop in CTX II as degradation product of type II collagen positive against degradation of extracellular matrix positive against degradation of extracellular matrix
Abubaker et al., 1996 Okuda et al., 2000
no
no
Yasuoka et al., 2000 Yasuoka et al., 2000 Oestergaard et al., 2006 Oestergaard et al., 2006 Ganesan et al., 2008 Gao and Du, 2008
no
no
yes
yes
yes
yes
yes
yes
yes
Table 4. (Continued ) Species Hormone replacement Result Estrogen deciency harmful? yes no Improvement with estrogen replacement? Improvement with estrogen receptor modulator? Author
mouse (OVX) STR/ort mouse (OVX)
DBA/1 mouse (OVX)
raloxifen
mouse (OVX + meniscus destabilization)
rabbit (OVX) rabbit (OVX + meniscectomy) rabbit (OVX)
17-E2 tamoxifen 17-E2 17-E2 intraarticular
collapse of articular cartilage strain spontaneously develops OA. no increase in OA due to OVX reduction of cartilage oligomeric matrix protein (COMP) as degradation product of type II collagen. TNF- reduced. meniscus-destabilized mice develop more OA than mice with meniscus destabilization + OVX exacerbation of OA improvement of OA damage to articular cartilage, up-regulation of ER cartilage damage high level of damage to articular cartilage
Da Silva et al., 1998 Chambers et al., 2001
yes
Jochem et al., 2007
likely not
Ma et al., 2007
no yes
yes -
Rosner et al., 1982b Rosner et al., 1982b Tsai and Liu, 1992a,b Tsai and Liu, 1993 Castaneda et al., 2010
rabbit (OVX) rabbit (bilateral OVX)
yes
no -
269
270
Table 4. (Continued ) Species Hormone replacement Result Estrogen deciency harmful? Improvement with estrogen replacement? yes Improvement with estrogen receptor modulator? Author
cows (OVX)
17-estradiol
sheep (OVX) monkey (OVX)
17-E2
monkey (OVX)
17-E2
monkey (OVX) Minipig (OVX)
17-E2 -
Minipig (OVX)
improved uptake of articular cartilage blocks into surrounding tissues exacerbation of biomechanical properties raised synovial level of insulin-like growth factor I and II (IGF-I and II) as well as insulin-like growth factor binding protein-1 and 3 (IGFBP-1 and 3) increased proteoglycan synthesis. more insulin-like growth factor binding protein-2 (IGFBP-2) reduction of osteoarthritic lesions change in relation of low to high sulfated glycosaminoglycans reduced glycosaminoglycan content
Englert et al., 2006
yes -
yes
Turner et al., 1997 Fernihough et al., 1999
yes
Richmond et al., 2000
yes
yes -
Ham et al., 2002 Claassen et al., 2002 Claassen et al., 2006a
yes
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5.7.3. Castration Elimination of male hormones by means of orchiectomy prevents development of degenerative articular diseases in mice of the DBA/2JN strain (Sokoloff et al., 1965). Castrated mice of the C57B1 strain develop a gonarthrosis that is improved by administration of -estradiol benzoate (Silberberg and Silberberg, 1969). In a study with male mice with OA induced by meniscus destabilization, orchiectomized animals develop less severe OA than normal animals (Ma et al., 2007). Administering dihydrotestosterone to orchiectomized mice exacerbates the OA. In ovariectomized cynomolgus monkeys, in which an age-dependent OA develops similar to what is observed in humans, administration of testosterone has no inuence on disease severity (Carlson et al., 1994). 5.7.4. Summary: inuence of articially induced androgen deciency Conclusions can only be drawn with a good measure of caution from animal experiments with articially induced androgen deciency, since a mouse model was used in most studies (Table 5). In mice, an orchiectomy-induced androgen deciency tends to improve articular cartilage status. 5.8. Clinical studies on the inuence of estrogen replacement therapy or estrogen/progesterone combination therapy 5.8.1. No inuence or negative inuence of estrogen replacement therapy The Framingham Osteoarthritis Study investigated whether estrogen replacement therapy initiated at or after menopause is associated with a change in the incidence of gonarthrosis. No signicant inuence on the risk of later occurrence of radiologically diagnosed osteoarthritis was determined (Felson, 1990). In the Ulm Osteoarthritis Study involving 475 women who had received a hip or knee joint endoprosthesis, no evidence was seen of a protective effect of estrogen replacement therapy in articular cartilage (Erb et al., 2000). In a study involving 1,001 postmenopausal women, estrogen replacement therapy was associated with an increased risk of development of coxarthrosis (von Mhlen et al., 2002). Only a slight correlation was determined between an estrogen deciency and articular cartilage changes in postmenopausal women based on immunohistochemical detection of the cartilage degradation product CTX-II, (Bay-Jensen et al., 2009). A study involving postmenopausal Chinese women provided evidence to the effect that metabolites of estrogen metabolism, in particular raised levels of 2-hydroxyestradiol, may play a role in the pathogenesis of OA (Gao et al., 2010). 5.8.2. Positive inuence of estrogen replacement therapy A study involving 539 postmenopausal American women revealed that estrogen replacement therapy lasting 10 years or longer tends to reduce, albeit not signicantly, the risk of mild to severe hip joint arthrosis symptoms (Nevitt et al., 1996). A study involving 76 patients suffering from osteoarthrosis deformans revealed gender differences in that the women showed a dysbalance of sex hormones with hyperestrogenemia and hyperprogesteronemia (Neiko et al., 1996). In treatment of the osteoarthrosis deformans with sex hormones (synestrol, testosterone), local treatment with ultraphonophoresis proved supe-
272 H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293
Table 5. Animal models with articially induced androgen deciency due to orchiectomy (castration, ORCH). Species Hormone replacement 17-E2 Result Androgen deciency harmful? Improvement with androgen replacement? Improvement with estrogen replacement? no Author
rat (ORCH)
rat (ORCH)
dihydrotestosterone
DBA/2JN mouse (ORCH) C57B1 mouse (ORCH)
-estradiol benzoate -
mouse (ORCH + meniscus destabilization) mouse (ORCH + meniscus destabilization)
dihydrotestosterone
reduction of proteins in discus articularis des mandibular joint slows loss of extracellular articular cartilage matrix reduced development of OA gonarthrosis is improvement by -estradiol benzoate Orchiectomized animals develop milder cases of OA than normal animals upon application of dihydrotestosterone exacerbation of OA
Abubaker et al., 1996 Ganesan et al., 2008 Sokoloff et al., 1965 Silberberg and Silberberg, 1969 Ma et al., 2007
yes
no -
yes
no
no
Ma et al., 2007
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rior to ultrasonic treatment alone (Sabadyshin et al., 1988). In the Chingford Study with 644 women, a non-signicant protective effect of estrogen replacement therapy against development of osteophytes in the knee joint was observed (Hart et al., 1999). In a study with 81 women, those undergoing estrogen replacement therapy had larger knee joint cartilage volumes compared to untreated women (Wluka et al., 2001). In a study of 42,462 Italian women with an average age of 53 years, the risk of OA was reduced under hormone replacement therapy (Parazzini, 2003). Hanna et al. (2004) nd evidence in their review that estrogens are inuential factors in healthy joints. Reginster et al. (2003) arrive at the opposite conclusion concerning the risk, i.e. that primary estrogen replacement therapy cannot be recommended in cases of OA. 5.8.3. Summary: inuence of estrogen replacement therapy To sum up, in 9 clinical studies of the efcacy of an estrogen replacement therapy, no protective effect was observed in 4, a non-signicant protective effect was observed in 2 and a signicant protective effect was observed in 3 studies (Table 6). Of the studies showing a positive effect, one was based on a study with Italian women, all others involved Chinese women. It must be remembered that different population groups have different nutritional habits (olive oil, soya) and may react differently to estrogen replacement therapy. For example, a diet during youth containing phytoestrogens could inuence the concentration of estrogen receptors in later life or cause relevant epigenetic changes that inuence the OA risk. In view of the varying preconditions of the clinical study, it would appear that estrogen replacement therapy does not necessarily protect against OA. 5.8.4. Estrogen/progesterone combination therapy Data are also available on combination estrogen/progesterone hormone replacement therapies. In a study involving 969 postmenopausal American women with an average age of 66 years (Heart and Estrogen/Progestin Replacement Study, HERS), replacement therapy lasting 4 years with a combination of estrogens and progesterone did not have any signicant effect on knee joint pain (Nevitt et al., 2001). By contrast, a similar combination therapy in 65 postmenopausal Chinese women aged 45-75 years showed a mitigating inuence of the symptoms of knee joints arthrosis (Song et al., 2004). 5.9. Clinical studies on the inuence of testosterone on articular cartilage In a study involving 45 men in decades 5 to 6, the serum testosterone level showed a positive association with the cartilage volume of the medial tibial plateau (Cicuttini et al., 2003).
6. Sex hormones and cytokines

Mineralization of the laryngeal cartilages differs in men and women. The concentration of VEGF increases with age in women and decreases with age in men (Pufe et al., 2004b). The response of growth joint chondrocytes to administration of 17-estradiol can be modulated by transforming growth factor (TGF)- (Nasatzky et al., 1999). 17-estradiol and testos-
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Table 6. Clinical studies on the inuence of 17-estradiol on articular cartilage. Study Estrogen replacement therapy yes, beginning at onset of menopause of after menopause yes Patients Result Estrogen replacement therapy protective? no Author
Framingham Osteoarthritis Study -
539 postmenopausal American women
Chingford Study
yes
644 women
Ulm Osteoarthritis Study Heart and Estrogen/Progestin Replacement Study -
yes
yes
475 women with hip or knee joint endoprosthesis 969 postmenopausal American women 81 women
no inuence on risk of later radiologically diagnosed OA estrogen replacement therapy for 10 years or longer shows trend to reduced risk of hip joint arthrosis no signicant protective effects of estrogen replacement therapy on development of osteophytes in the knee joint no point of reference for protective effect on articular cartilage average age 66 years
Felson, 1990
yes, but not signicantly
Nevitt et al., 1996
yes, but not signicantly
Hart et al., 1999
no
Erb et al., 2000
no, no effect on knee joint pains yes
Nevitt et al., 2001
yes
treated women showed greater cartilage volume in knee joint compared to untreated women
Wluka et al., 2001
Table 6. (Continued ) Study Estrogen replacement therapy yes yes Patients Result Estrogen replacement therapy protective? no yes Author
1001 postmenopausal women 42462 Italian women
yes
65 postmenopausal Chinese women
greater risk of development of OA women at average age of 53 years showed reduced risk for OA average age 45 to 75 years
Von Mhlen et al., 2002 Parazzini, 2003
yes, slight alleviation of symptoms of knee joint arthrosis
Song et al., 2004
275
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terone interact with the proinammatory cytokine interleukin (IL) -1 by signicantly raising the level of synthesis of IL-6 (Guerne et al., 1990). IL-6 functions as a regulator in inammatory and immunological processes in synovia and articular cartilage and has been detected in the articular uid of persons with arthrosis. In pregnant rabbits, under the inuence of estrogens and relaxin, a reduced expression of tumor necrosis factor (TNF)-, a cytokine that is catabolic for articular cartilage, is detected (Hellio le Graverand et al., 1998). In rabbit articular cartilage cells, 17-estradiol suppresses the IL-1-induced nitrogen monoxide (NO) production, whereby the IL-1-induced enzyme nitrite synthetase is inhibited at the protein level (Richette et al., 2007). Under stimulation with the catabolic cytokine TNF-, incubation with 17-estradiol reduces the secretion of MMP-1 in osteoarthritic chondrocytes of postmenopausal female patients (Lee et al., 2003). In the same culture model, 17-estradiol does not exert any effect on expression of MMP-3, MMP-13 and TIMP-1 under stimulation with Il-1 or TNF- (Lee et al., 2003). Without cytokine stimulation, secretion of MMP-1 is signicantly reduced by 17-estradiol (Lee et al., 2003). Cytokines are differentiated in the cartilage as anabolic (growth enhancing) or catabolic (proinammatory or cartilage-degrading).
6.1. Cartilage-degrading (catabolic) cytokines 6.1.1. Interleukin-1beta (IL-1) and tumor necrosis factor-alpha (TNF-) IL-1 is classied as a catabolic cytokine. It has a proinammatory effect and contributes to the pathogenesis of OA (Shakibaei et al., 2007; Elshaier et al., 2009). IL-1 inhibits the mRNA expression of type II collagen (Frazer et al., 1994). Inhibition of complex I in mitochondria reduces, together with lowered adenosine triphosphate (ATP) production, the proteoglycan content of the extracellular matrix (Lpez-Armada et al., 2006). The mRNA expression of galactose-beta-1.3 glucuronosyltransferase, a key enzyme in glycosaminoglycan synthesis, is also inhibited (Gouze et al., 2001). Activation of the transcription factor NF-B, together with the inuence of IL-1, results in increased production of MMP-3 and 13 (Schulze-Tanzil et al., 2004; Ahmad et al., 2007), followed by degradation of extracellular matrix. Finally, IL-1 also induces a rise in the level of the enzyme cyclooxygenase-2 (COX-2), which is responsible for production of the prostaglandins that contribute to the pathogenesis of OA (Lyons-Giordano et al., 1993). da Silva et al. (1993a,b) demonstrated in female mice and male rodents, post ovariectomy or orchiectomy, that estradiol and androgens have a protective effect on inammatory degradation of extracellular matrix and are thus immunoregulatory agents. As a catabolic cytokine, TNF- contributes to the dysregulation of cartilage remodeling in OA (Koike, 2006). This cytokine inhibits the proteoglycan synthesis of chondrocytes (Saklatvala, 1986) and reduces the expression of type II collagen and aggrecan at the mRNA level (Squin and Bernier, 2003; Klooster and Bernier, 2005). The fact that stimulation with TNF- reduces ATP production in human chondrocytes also contributes to reduction of the proteoglycan content of the extracellular matrix of articular cartilages (Lpez-Armada et al., 2006). As a proinammatory cytokine, it contributes to cartilage degradation by inducing MMP-13 (Liacini et al., 2003). TNF- stimulates the mRNA expression of MMP-1, 3 and 13 in equine chondrocytes (Richardson and Dodge, 2000).
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Furthermore, TNF- also raises production of the binding protein for insulin-like growth factor (IGFBP-3), thus reducing the access of IGF-I to chondrocytes (Olney et al., 1995). This effect contributes to reduction of matrix by chondrocytes. Finally, TNF- induces the enzyme cyclooxygenase (COX)-2, which contributes to production of prostaglandins, at the mRNA level (Geng et al., 1995). The damaging effects on articular cartilage of TNF- can be prevented in vitro by inhibition of the signal transduction protein ERK (Djouad et al., 2009). 6.1.2. Vascular endothelial growth factor (VEGF) Recent studies have demonstrated that VEGF is among the degrading factors for articular cartilage. Normal articular cartilage is avascular and is nurtured via diffusion from the synovial uid. Osteoarthritic cartilage, in contrast to normal cartilage, is characterized by a reduced resistance to vascular invasion (Smith et al., 2003). Hypoxia and stimulation with IL-1 result in production of VEGF in human articular cartilage cells by way of activation of the MAP kinase signal transduction chain (Murata et al., 2006). The oxygen radicals resulting from every cellular reaction can also induce VEGF. Fay et al. (2006) experimentally induced immortalized chondrocytes of cell line C-28/I2 and explants of knee joint cartilage to produce radicals (reactive oxygen species, ROS), whereupon levels of both VEGF and the VEGF receptors VEGFR-1 and 2 increased. In an OA model induced in rabbits, the VEGF receptor VEGFR-2 played a particularly prominent role in progression of the disease (Zhou et al., 2009). VEGF contributes to cartilage-destructive processes within the framework of OA because this cytokine increases production of MMPs (Enomoto et al., 2003) and induces the cartilage-destructive cytokines IL-1 and TNF- (Pufe et al., 2004a). IL-1-stimulated chondrocytes possess a functional complex made up of beta(1)integrin and VEGF receptor 3 (VEGFR-3). beta(1)-integrins are themselves connected to other cell surface receptors that can initiate destructive processes in OA (Shakibaei et al., 2003). In the immortalized chondrocyte cell lines C-28/I2 and T/C-28a2, a VEGF stimulation raises secretion of MMP 1, 3 and 13 with reduced expression of TIMP-1 and 2 (Pufe et al., 2004a). Raised concentrations of VEGF were measured in osteoarthritic cartilage, whereby splice variant VEGF 121 diffuses freely and splice variant 189 binds to extracellular proteoglycans (Pufe et al., 2001). Both proteins contribute to inammation, chemotaxis of macrophages and angiogenesis by means of autocrine and paracrine secretion of chondrocytes. 6.1.3. Antimicrobial peptides (AMP) Articular cartilage and synovial membrane, as well as immortalized chondrocytes of cell line T/C-28a2 are capable under normal and pathogenic conditions of producing antimicrobial substances and antimicrobial peptides (APs) (Paulsen et al., 2001, 2002; Varoga et al., 2004). Septic synovial membranes, and synoviocytes cultured in contact with bacteria, produce larger amounts of APs than in a healthy state (Varoga et al., 2009). Lysozyme, lactoferrin and secretory phospholipase A2 (sPA2 ) are considered antimicrobial substances; APs are human neutrophilic alpha-defensins 1-3 (HNP 1-3) and human beta-defensins 2 and 3 (HBD-2 and HBD-3). Healthy chondrocytes react to exposure to gram-negative bacteria with increased expression of HBD-2 (Varoga et al., 2004). Catabolic cytokines, for example TNF- and IL-1, cause pronounced induction of HBD-3 in cultured chondrocytes (Varoga
278
et al., 2005b). HBD-2, expression of which is regulated by TNF-, IL-1 and IL-6, plays a key role in osteoarthritic cartilage (Varoga et al., 2006). 6.1.4. Trefoil factor family peptide 3 (TFF 3) Trefoil factor family peptide 3 (TFF3), also known as intestinal trefoil factor (ITF), has numerous functions (Hoffmann, 2005). In the pancreatic islets it is expressed together with insulin and there contributes to cell adhesion and migration (Jackerott et al., 2006). It has also been demonstrated that TFF3 is regulated by estrogens (May et al., 2003). In mucosa, TFF3 alters the viscosity of the mucus, has a motogenic effect, supports epithelial regeneration and counteracts apoptosis (Hoffmann, 2007). On the surface of the eye, TFF3 is induced within the framework of inammations (herpes keratitis) (Steven et al., 2004). It also supports corneal regeneration and corneal wound healing (Paulsen et al., 2008). As in the cornea, TFF3 is only induced in articular cartilage under pathological conditions, e.g. OA or a bacterial joint infections, in vitro and in vivo; here, in contrast to the eye surface, the peptide enhances the degradation of extracellular matrix by up-regulation of MMP 1, 3 and 13 and has a proapoptotic effect (Rsler et al., 2010). Mice that do not express TFF3 develop an accelerated presbyacusis (Lubka et al., 2009). Examinations of the cochleae of these mice reveal participation of TFF3 in the apoptotic process. 6.2. Cartilage-synthesizing (anabolic) cytokines 6.2.1. Insulin-like growth factor-I (IGF-I) The anabolic cytokine IGF-I is an important factor in preservation of articular cartilage (Franchimont and Bassleer, 1991). IGF-I stabilizes the chondrogenic potential of chondrocytes (Schmidt et al., 2006; Shakibaei et al., 2006). 85% of the intracellular domain of the IGF-I receptor coincides with that of the insulin receptor. IGF-I activates both the IGF-I receptor and the insulin receptor and raises the level of synthesis of proteoglycan, which is responsible for cartilage elasticity, by stimulating phosphoinosite-3-kinase (Starkman et al., 2005). Genetically altered chondrocytes, which express IGF-I, accelerate wound healing in articially induced articular cartilage defects, whereby the expression of the cartilagespecic type II collagen is increased approx. 100-fold (Goodrich et al., 2007). It was also demonstrated in cultured bovine articular cartilage cells that application of insulin signicantly increase synthesis of type II collagen (Claassen et al., 2006c). An IGF-I deciency is responsible for the degree of severity of osteoarthritic lesions (Ekenstedt et al., 2006). IGF-I is the cartilage matrix degrading enzyme that down-regulates the IL-1-stimulated mRNA expression of MMP1, -3, -8 and -13 in human chondrocytes (Hui et al., 2001). The binding proteins of insulin-like growth factor (IGFBPs), control the distribution of IGF-I between the extracellular space and cellular compartment and alter the bioactivity of IGF-I by modulating the IGF-I receptor (Scheidegger et al., 2000). Experiments with monkey and rat chondrocytes revealed a signicant reduction of the IGF-I response parallel to increasing age (Martin et al., 1997; Loeser et al., 2000). IGF-I signals are mediated via the phosphatidylinositol 3 -kinase (PI3K)/Akt signal transduction path. A raised expression of the Akt inhibitor TRB3 in OA may be responsible for the weakened IGF-I reaction in this disease (Cravero et al., 2009). Age-related increase in IGFBPs results in a reduction of
279
IGF-I-stimulated proteoglycan synthesis (Okuda et al., 2001). In women with coxarthrosis, the serum IGF-I level is signicantly lower than in healthy women (Lis et al., 2005). 6.2.2. Transforming growth factor-beta (TGF-) TGF-, which is classied as an anabolic cytokine, plays a role in cartilage regeneration (Blaney Davidson et al., 2005; Roman-Blas et al., 2007). The synthesis of type II collagen and proteoglycans, important components of the extracellular matrix of articular cartilage, is enhanced by TGF- (Frazer et al., 1994; Iqbal et al., 2000). TGF-3 in particular has an anabolic effect on the expression of the 1-collagen II gene in chondrogenesis (Tang et al., 2009). In the proteoglycan metabolism of murine articular cartilage cells, TGF- and IGF-I function synergetically, which is reected in enhanced mRNA expression of aggrecan, the main proteoglycan of articular cartilage (Tsukazaki et al., 1994). TGF- up-regulates the expression of TIMP-1, an antagonist of the cartilage-degrading MMPs (Gnther et al., 1994; Frenkel et al., 2000). TGF- induces production of TIMP-3, which protects the articular cartilage matrix from degradation just as TIMP-1 does, by activating the ERK/MAPK signal transduction path (Qureshi et al., 2005). In cultured rabbit chondrocytes, application of TGF-1 raises the level of collagen II and glycosaminoglycan synthesis (Ab-Rahim et al., 2008). 6.3. Summary: inuence of the interaction of sex hormones and cytokines The brief overview shows that sex hormones interact with various cytokines in articular cartilage metabolism (Fig. 5a, b). For instance, 17-estradiol and testosterone interact with IL-1. During pregnancy, the inuence of estrogens and relaxin result in a reduced expression of the catabolic cytokine TNF-. Various catabolic cytokines contribute to articular cartilage metabolism without interacting with hormones (Fig. 5a, b). Cytokines can also interact with transcription factors and enzymes. For instance, IL-1 activates synthesis of MMP-3 and 13 by switching on NF-kB and TNF- induces the enzyme COX-2. VEGF also increases the secretion of MMP 1, 3 and 13 and at the same time induces IL-1 and TNF-. The role of antimicrobial peptides beyond the purely antimicrobial function has received little research attention to date, for example HBD-2, which can also be induced by the catabolic cytokines TNF- and IL-1 (Fig. 6). TFF-3 is apoptotic in articular cartilage, quite a contrast to the effect ascribed to this substance in the mucosa. The anabolic cytokines should be kept in mind in the search for new therapeutic substances and approaches to improve articular cartilage symptoms. TGF-1, for instance, raises levels of the collagen II and glycosaminoglycan synthesis.
7. Cartilage-degrading enzymes
A balance between MMPs and TIMPs, the regulatory proteins of catabolic collagen metabolism, protects the collagen brils of the extracellular matrix of articular cartilage from non-physiological degradation. Numerous studies conrm that this balance is destroyed in OA (Dean et al., 1989; Dean, 1991; Greenwald, 1994; Martel-Pelletier et al., 1994). Compared to healthy cartilage, arthritic tissue is characterized by increased degradation of
280
type II collagen (Billinghurst et al., 1997). MMP 8 and 13 enzymes chiey responsible for degradation of type II collagen in the articular cartilage of the rat (Ando et al., 2009). Articial immobilization of the knee joint in these animals results in increased expression of MMP-8 and 13 by the hypertrophic chondrocytes of the deep articular cartilage. Incubation of primary articular cartilage cells from female patients post hip or knee joint surgery cultured in an alginate system with 17-estradiol unfolds a protective effect by reducing the expression of MMP-1, 3 and 13 (Claassen et al., 2010a). The enzyme cyclooxygenase-2 (COX-2) catalyzes the reaction that produces prostaglandin E (2) from arachidonic acid, which latter is classied as an inammatory mediator in OA (Fermor et al., 2002). A raised prostaglandin E (2) level of within the framework of OA is accompanied by raised levels of proteoglycan degradation (Hardy et al., 2002). In a pregnant rabbit animal model, a reduced expression of COX-2 is observed under the inuence of estrogens and relaxin (Hellio le Graverand et al., 1998). 7.1. Summary: inuence of cartilage-degrading enzymes Sex hormones presumably act upon other enzymes that contribute to articular cartilage metabolism. The most recent results obtained by our working group, showing that 17estradiol inhibits the expression of the articular cartilage matrix-degrading enzymes MMP1, 3 and 13, are only the beginning of a series of new ndings (Fig. 6).
8. Estrogens and insulin

8.1. Inuence of insulin Insulin exerts a pronounced effect on the differentiation of cartilage precursor cells into chondroblasts and chondrocytes in the growth joint (Maor et al., 1993). Insulin also enhances the uptake of {3 H}-thymidine and {35 S}-sulfate in physiological concentrations (Maor et al., 1993). Administration of insulin also signicantly increases the incorporation of proline and synthesis of type II collagen by articular cartilage cells (Claassen et al., 2006c). By contrast, 17-estradiol alone does not signicantly inuence type II collagen synthesis. Surprisingly, preincubation of the cells with physiological doses of 17-estradiol results in a signicant suppression of the protein anabolic effect of insulin on proline incorporation and type II collagen synthesis (Claassen et al., 2006c). Preincubation of the cells with 17-estradiol and tamoxifen (an estrogen receptor antagonist with partial intrinsic activity) or with 17-estradiol and ICI 182.780 (a full estrogen receptor antagonist) reverses the suppression of the stimulatory effect of insulin (Claassen et al., 2006c). These ndings support mediation of the effect via estrogen receptors; they also provide important evidence that 17-estradiol contributes to the mechanism of action of insulin at chondrocytes and, under certain conditions, even contributes to a reduction of insulin sensitivity (Fig. 6). Application of physiological doses of 17-estradiol does not increase the uptake of radiolabeled sulfate in bovine articular cartilage cells. Furthermore, physiological doses of 17-estradiol have no inuence on histochemically evaluated glycosaminoglycan synthesis, whereby supraphysiological doses inhibit it (Fig. 11). In summary: 17-estradiol does not
281
Fig. 11. Histochemical staining of glycosaminoglycans (GAGs) in female bovine articular chondrocytes cultured on coverslips and incubated without (a) or with 109 M (b), 107 M (c) and 104 M (d) 17-estradiol. GAGs are stained blue a,b: Compared to control, extracellular matrix of chondrocytes incubated with a physiological dose of 109 M 17-estradiol show no more pericellular blue staining for GAGs. c,d: By contrast, high doses of 107 M-104 M 17-estradiol reveal diminished staining for GAGs in comparison to the control (a). Concomitantly, fewer chondrocytes surrounded by a blue rim of GAGs are observed when compared with the control (a). It can be concluded that supraphysiological doses of 17-estradiol impede GAG synthesis. a-d: alcian blue. Nuclei of cells were counterstained with nuclear fast red. a-d: bar 35 m.
act in isolation. The phytoestrogens daidzein and genistein, estrogen-like substances from plants, also show no signicant inuence for instance on the uptake of radiolabeled sulfate in bovine chondrocytes when applied alone (Claassen et al., 2008; Fig. 6). In contrast to this, insulin increases sulfate uptake in bovine chondrocytes signicantly. Interestingly enough, preincubation of chondrocytes with daidzein or genistein results in a signicant increase in the anabolic effect of insulin on sulfate uptake; when the cells are preincubated with 17-estradiol, sulfate uptake remains at the level induced by insulin alone (Claassen et al., 2008). Since sulfate update can be considered a marker for proteoglycan synthesis, the potentiating effect of the two phytoestrogens on sulfate uptake must be considered benecial for the articular cartilage. It is notable that this potentiating effect also occurs within a broad spectrum of dosage of daidzein and genistein (1011 M-105 M).
282
8.2. Osteoarthritis and diabetes mellitus Insulin exerts a direct stimulatory effect on extracellular matrix synthesis, in view of the fact that cultured articular cartilage shows a pronounced increase of proteoglycan production when insulin is applied (Maor et al., 1993). A disturbance of the glucose metabolism is extremely harmful to articular cartilage, as demonstrated by a small number of studies that have apparently been ignored with regard to OA therapy. Degenerative changes in the articular cartilage can be caused in OA by metabolic disturbances of the glucose balance, such as occur in more dramatic form in diabetes mellitus (Mobasheri et al., 2002). The insulin receptor has been detected at the mRNA and protein levels on immortalized chondrocytes from the cell lines C-28/I2 and T/C-28a2 as well as on primary human chondrocytes (Claassen et al., 2010b). It is known from electron microscopic investigations of mice (Silberberg et al., 1968) and rats (Niethard, 1986) with articially induced diabetes mellitus that chondrocytes react clearly to a disturbed glucose metabolism. The results include cell necrosis, changes in cell organelles in the form of swollen mitochondria and a reduction of the endoplasmic reticulum as well as reduced intracellular glycogen storage. The hexosamine content of the articular cartilage was also reduced in diabetic rats (Niethard, 1986). Rats with induced diabetes mellitus also show reduced collagen production in the articular cartilage two weeks after diabetes induction, correlating with the circulating IGF-I level (Spanheimer et al., 1988; Umpierrez et al., 1989). In combination with the growth and differentiation factor 5 (GDF5), insulin increases type II collagen synthesis in primary human articular cartilage cells (Appel et al., 2009). It has also been known for some time that diabetic children show stunted growth (Birkbeck, 1972). More recent studies of diabetic patients have revealed a softer consistency, as well as altered biomechanical properties, of the articular cartilage (Athanasiou et al., 1999). 8.3. Summary: inuence of insulin When summarized, the results show clearly that the signicance of insulin in articular cartilage metabolism has been underestimated (Fig. 6). Insulin signicantly raises the levels of collagen II synthesis and uptake of radiolabeled sulfate a marker for proteoglycan synthesis. How both 17-estradiol and the phytoestrogens daidzein and genistein inuence the signal transduction path of the insulin receptor is not known (Fig. 6). An objective of our future efforts will therefore be an analysis of the interrelationships between sex hormone and insulin signal transduction paths in relation to articular cartilage metabolism.
9. Concluding remarks
It is clear from the ndings described here that success in osteoarthritis therapy to date has been almost completely restricted to surgical success. No methods have been devised to date for early recognition and prevention of articular diseases, especially in postmenopausal women. This is partly due to a lack of understanding of the metabolism of chondrocytes, in particular the inuence of hormonal changes in the middle period of life (menopause,
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andropause). The pill has been available for family planning since the 1960s. Estrogen receptor modulators such as tamoxifen are now used in therapy of breast cancer. Unpleasant symptoms experienced during the climacteric period were until recently treated with generous and long-term estrogen replacement therapies. On adverse effect of this hormonal therapeutic concept has been an accumulation of estrogens in the environment, in particular in the groundwater (Tyler et al., 2009). For this reason as well, it is important to arrive at an understanding of whether estrogens damage or benet cartilage tissue. It is of signicance in this context that bisphenol, a so-called endogenous disruptor derived from industrial plastic wastes, blocks the cartilage-protective effect of 17-estradiol, which is based on downregulation of the transcription factor NF-k B (Wang et al., 2010). The long-term objective must be to gain an understanding of the relevant changes in articular cartilage metabolism so that preventive approaches to OA can be established that will delay joint endoprosthetic measures for as long as possible. Hormonal agents, such as have already been used with success in treatment of mammary carcinoma, could also be helpful in postmenopausal osteoarthritis. It must be remembered, however, that while some postmenopausal women show a tendency to osteoarthritis, others develop osteoporosis. A short-term hormone replacement or estrogen receptor modulation therapy may, if benecial, have to begin before the menopause. In cases in which short-term classic hormone replacement therapy is contraindicated, treatment with phytoestrogens could offer an alternative. Acknowledgments We would like to thank Jrg Pekarsky for drawing most of the gures and Michael Beall for the English. We acknowledge support by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), Bonn, Germany (program grant nos.: PA738/6-1, PA738/91, PA 738/9-2, BR3681/2-1), the Wilhelm Roux program, Halle, Germany (program grant nos.: FKZ 13/17, FKZ 09/17, FKZ 14/24, FKZ 14/25, FKZ 20/31), and the GI-Company Inc., Framingham, USA. References
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PROGRESS IN HISTOCHEMISTRY AND CYTOCHEMISTRY

Accepted 21 October 2010

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

4. Sex hormones in cartilage

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

Claassen et al., 2001 Priest and Koplitz 1966

female rat rabbit rabbit rabbit

animal model cell culture cell culture cell culture

negative negative positive negative

rabbit female rabbit

cell culture cell culture

Itagane et al., 1991

Tsai et al., 1998 Hellio le Graverand et al., 1998

17-estradiol: neutral, positive on collagen II synthesis, positive on glycosaminoglycan synthesis

Ab-Rahmin et al., 2008

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

neutral stabilization of plasma membrane

Mackintosh and Mason, 1988 Claassen et al., 2005

Claassen et al., 2006

17-estradiol: neutral, daidzein: positive, genistein: positive negative negative positive

Claassen et al., 2008

human adipose women women

cell culture clinical trial cell culture

Postmenopausal women women

estrogen replacement therapy cell culture

Wluka et al., 2004

DNA synthesis, uptake of radiolabeled sulfate, activity alkaline phosphatase

Kinney et al., 2005

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

women, immortalized cell lines C-28/I2 and T/C-28a2

cell culture, RT-PCR, Western Blot

Claassen et al., 2010b

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

5. Sex hormones and osteoarthritis

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

male rats female and male rabbit rabbit

animal model cell culture cell culture

Age-dependence preincubation with testosterone, further incubation with IGF-I -

cell culture animal model

Mackintosh and Mason, 1988 Englert et al., 2006

Franchimont and Basler 1991 Hanna et al., 2007

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

264 H. Claassen et al. / Progress in Histochemistry and Cytochemistry 45 (2011) 239293

Colombo et al., 1983

Rosner et al., 1986