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Extracellular matrix changes in knee joint cartilage following bone-active drug treatment
Abstract
Certain drugs or treatments that are known to affect bone quality or integrity might have side effects on the extracellular matrix of articular cartilage. We investigated the effects of vitamin D and calcium deficiency, estrogen deficiency, and hypercortisolism alone or in combination with bisphosphonates or sodium fluoride in an animal model, viz., the Göttingen miniature pig (n=29). The articular cartilage from knee joints was analyzed for its content of glycosaminoglycans (GAGs, as macromolecules responsible for the elasticity of articular cartilage) by a spectrometric method with dimethylene blue chloride. In cryo- or paraffin sections, alkaline phosphatase (AP, as an enzyme indicating mineralization or reorganization of articular cartilage matrix) was localized by enzyme histochemistry, and positive cells were counted, whereas differently sulfated GAGs were stained histochemically. A significant decrease in GAG content was measured in ovariectomized and long-term glucocorticoid-treated animals compared with untreated animals. In the glucocorticoid/sodium fluoride group, GAGs were significantly diminished, and significantly fewer AP-positive chondrocytes were counted compared with the control. GAG content was slightly higher, and significantly more AP-positive chondrocytes were counted in short-term glucocorticoid-treated animals then in the control group. GAGs, as part of proteoglycans, are responsible for the water-storage capacity that gives articular cartilage its unique property of elasticity. Thus, ovariectomy and long-term glucocorticoid therapy, especially when combined with sodium fluoride, have detrimental effects on this tissue.
... Third, we used the rabbit model, and future studies might consider using a larger-animal model eg, the porcine model, which has been used to investigate aging 43 and menopause. 44,45 In fact, these studies in the porcine model investigated skeletalmuscle changes with aging 43 and bone and cartilage changes with menopause, 44,45 where calcium and vitamin D were evaluated due to hormonal and dietary influences. Fortunately, we have previously established a joint-laxity measurement in the porcine model and understand the advantages and disadvantages of comparing this model to humans. ...
... Third, we used the rabbit model, and future studies might consider using a larger-animal model eg, the porcine model, which has been used to investigate aging 43 and menopause. 44,45 In fact, these studies in the porcine model investigated skeletalmuscle changes with aging 43 and bone and cartilage changes with menopause, 44,45 where calcium and vitamin D were evaluated due to hormonal and dietary influences. Fortunately, we have previously established a joint-laxity measurement in the porcine model and understand the advantages and disadvantages of comparing this model to humans. ...
Background
When considering the “joint as an organ”, the tissues in a joint act as complementary components of an organ, and the “set point” is the cellular activity for homeostasis of the joint tissues. Even in the absence of injury, joint tissues have adaptive responses to processes, like aging and menopause, which result in changes to the set point.
Purpose
The purpose of this study in a preclinical model was to investigate age-related and menopause-related changes in knee-joint tissues with the hypothesis that tissues will change in unique ways that reflect their differing contributions to maintaining joint function (as measured by joint laxity) and the differing processes of aging and menopause.
Methods
Rabbit knee-joint tissues from three groups were evaluated: young adult (gene expression, n=8; joint laxity, n=7; water content, n=8), aging adult (gene expression, n=6; joint laxity, n=7; water content, n=5), and menopausal adult (gene expression, n=8; joint laxity, n=7; water content, n=8). Surgical menopause was induced with ovariohysterectomy surgery and gene expression was assessed using reverse-transcription quantitative polymerase chain reaction.
Results
Aging resulted in changes to 37 of the 150 gene–tissue combinations evaluated, and menopause resulted in changes to 39 of the 150. Despite the similar number of changes, only eleven changes were the same in both aging and menopause. No differences in joint laxity were detected comparing young adult rabbits with aging adult rabbits or with menopausal adult rabbits.
Conclusion
Aging and menopause affected the gene-expression patterns of the tissues of the knee joint differently, suggesting unique changes to the set point of the knee. Interestingly, aging and menopause did not affect knee-joint laxity, suggesting that joint function was maintained, despite changes in gene expression. Taken together, these findings support the theory of the joint as an organ where the tissues of the joint adapt to maintain joint function.
... The hormone probably stabilizes the plasma membrane of the articular cartilage cells. III: In the animal model ovariectomized Göttingen Minipigs, the glycosaminoglycan content in the knee joint is significantly reduced (Claassen et al., 2002(Claassen et al., , 2006a. IV: Catabolic cytokines, for example TNF-␣ and IL1-ß, cause a pronounced induction of human beta-defensin (HBD) 3 in cultured articular cartilage cells (Varoga et al., 2005a/b). ...
... It was demonstrated in the animal model ovariectomized and intact Göttingen 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 significantly 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 deficiency for the articular cartilage. ...
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 influence 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 an information matrix that identifies what is known, with supporting references, and identifies areas for further investigation.
... The effects of GCs on the GAG content in cartilage from knee joints were studied in Göttingen miniature pigs. Short-term prednisolone treatment at a dose of 1 mg/kg/day for 2 months and 0.5 mg/kg/day for 6 months did not affect GAG content in the cartilage, whereas long-term prednisolone treatment at a dose of 1 mg/kg/day for 2 months and then 0.5 mg/kg BW/day for 13 months resulted in a decrease in GAG content by 14% (p < 0.04) [33]. In a study involving multiple injections of betamethasone into the knee of Californian rabbits, only 6-8 injections with a 1-week interval significantly decreased total PG content in the articular cartilage from tibial plateaus, whereas 1-5 injections do not change the PC content [34]. ...
Glucocorticoids are steroid hormones that play diverse roles in numerous normal and pathological processes. They are actively used to treat a wide variety of diseases, including neurodegenerative and inflammatory diseases, cancers, and COVID-19, among others. However, the long-term use of glucocorticoids is associated with numerous side effects. Molecular mechanisms of these negative side effects are not completely understood. Recently, arguments have been made that one such mechanisms may be related to the influence of glucocorticoids on O-glycosylated components of the cell surface and extracellular matrix, in particular on proteoglycans and glycosaminoglycans. The potential toxic effects of glucocorticoids on these glycosylated macromolecules are particularly meaningful for brain physiology because proteoglycans/glycosaminoglycans are the main extracellular components of brain tissue. Here, we aim to review the known effects of glucocorticoids on proteoglycan expression and glycosaminoglycan content in different tissues, with a specific focus on the brain.
... Two main types of GAGs can be distinguished: unsulfated GAGs (hyaluronan) and sulfated GAGs (heparin and heparan sulfate, chondroitin sulfate, dermatan sulfate and keratan sulfate) (Gandhi and Mancera 2008). Bone GAGs consist of mainly chondroitin sulfate (CS) and small amounts of hyaluronan (HA) (Claassen et al. 2006;Salbach et al. 2012b) and were reported to undergo continuous changes during MSC differentiation but are also affected by aging (Grzesik et al. 2002) and pathologies such as type 2 diabetes mellitus, rheumatoid arthritis and systemic lupus erythematosus, where changes in GAG pattern also contribute to disease aggravation (Mania et al. 2009). ...
The bone microenvironment is a complex tissue in which heterogeneous cell populations of hematopoietic and mesenchymal origin interact with environmental cues to maintain tissue integrity. Both cellular and matrix components are subject to physiologic challenges and can dynamically respond by modifying cell/matrix interactions. When either component is impaired, the physiologic balance is lost. Here, we review the current state of knowledge of how glycosaminoglycans – organic components of the bone extracellular matrix – influence the bone micromilieu. We point out how they interact with mediators of distinct signaling pathways such as the RANKL/OPG axis, BMP and WNT signaling, and affect the activity of bone remodeling cells within the endosteal niche summarizing their potential for therapeutic intervention.
... Degenerative joint disease may be classified as primary, where no predisposing factors are present (mainly occurring in older animals) or secondary, associated with an underlying abnormality (malformation or deformation) in the joint or supporting structures leading to premature degeneration of the joint cartilage. 200,[206][207][208] Chondrocyte dysfunction leads to aberrant maintenance of the matrix in articular cartilage. The resulting alterations in the shape and continuity of cartilage surfaces permits the release of pro-inflammatory factors and promotes joint instability, thereby leading to secondary reactions in other joint components. ...
The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) Project ( www.toxpath.org/inhand.asp ) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in most tissues and organs from the minipig used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet ( http://www.goreni.org/ ). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.
... Other authors have investigated the effect of estrogen deficiency on the synthesis of glycosaminoglycans (GAG), which are an important component of the connective tissue. A significant decrease in GAG from knee joints was found in both cryo-and paraffin sections of OVX animals compared with sham controls, indicating that loss of estrogens has a detrimental effect on cartilage tissue [80]. ...
Hormone therapy (HT) has menopausal symptom control as the main indication. However, and because estrogens have receptors spread over many systems in the body, the use of hormones may have consequences on health. Hormone therapy consists of estrogens essentially, since menopausal symptoms mainly derive from estrogen deprivation at different target tissues. However, the normal ovarian cycle includes the regular secretion of progesterone during the days of the luteal phase. Progesterone has a neutralizing effect over endometrial proliferation, this being the reason for adding progestogens to HT formulations in order to protect endometrium. So, guidelines recommend that women with uterus use estrogen plus progestogens, which may be combined in different forms. And because of that, the impact of HT on health should also consider the specific effects of both estrogens and progestogens in addition to those of only estrogens. While this has turned to be of minor importance in some areas, it is not so in others, like for example the oncological risk.
... Moreover, a decrease of collagen II degradation products in the urine has been observed following estrogen application in ovariectomized rats [8]. Claassen et al. showed a statistically significant decrease in the GAG content in miniature pigs with an experimentally induced estrogen deficiency in comparison with nontreated animals [9]. Furthermore, collagen II synthesis was found to be increased when chondrocytes from the rib growth zone of female rats were incubated with estradiol, supporting the hypothesis that estradiol deficiency in postmenopausal women might be associated with a higher incidence of OA [10]. ...
The aim of this study was to investigate the biological effects of sex hormones (17 β -estradiol and testosterone) on rabbit articular chondrocytes from different genders. We cultured primary rabbit articular chondrocytes from both genders with varying concentration of sex hormones. We evaluate cell proliferation and biochemical functions by MTT and GAG assay. The chondrocyte function and phenotypes were analyzed by mRNA level using RT-PCR. Immunocytochemical staining was also used to evaluate the generation of collagen-II. This study demonstrated that 17 β -estradiol had greater positive regulation on the biological function and gene expressions of articular chondrocytes than testosterone, with the optimal concentrations of 10(-6) and 10(-7) M, particularly for female chondrocytes.
... Minipigs, unlike rats or sheep, develop glucocorticosteroid-induced osteoporosis (GIO) without the need for simultaneous ovariectomy [13]. We have shown earlier that GIO can be monitored by measurement of bone mineral density [11, 13] and is accompanied by changes in cartilage matrix [29] and bone tissue vascularisation [30]. ...
Calcium and vitamin D deficiency impairs bone health and may cause rickets in children and osteomalacia in adults. Large animal models are useful to study experimental osteopathies and associated metabolic changes. We intended to modulate vitamin D status and induce nutritional osteomalacia in minipigs. The control group (n = 9) was fed a semisynthetic reference diet with 6 g calcium and 6,500 IU vitamin D3/kg and the experimental group (n = 10) the same diet but with only 2 g calcium/kg and without vitamin D. After 15 months, the deficient animals were in negative calcium balance, having lost bone mineral density significantly (means ± SEM) with -51.2 ± 14.7 mg/cm(3) in contrast to controls (-2.3 ± 11.8 mg/cm(3)), whose calcium balance remained positive. Their osteoid surface was significantly higher, typical of osteomalacia. Their plasma 25(OH)D dropped significantly from 60.1 ± 11.4 nmol/L to 15.3 ± 3.4 nmol/L within 10 months, whereas that of the control group on the reference diet rose. Urinary phosphorus excretion and plasma 1,25-dihydroxyvitamin D concentrations were significantly higher and final plasma calcium significantly lower than in controls. We conclude that the minipig is a promising large animal model to induce nutritional osteomalacia and to study the time course of hypovitaminosis D and associated functional effects.
... It is well established that GAGs are responsible for the water-storage capacity that gives articular cartilage elasticity. In normal bone, GAG's decrease because of age, therapy, or hormonal imbalance (17). On the other hand, elevated GAG levels have been shown to contribute to the worsening of the condition of patients suffering from autoimmune conditions such as rheumatoid arthritis and systemic lupus erythematosus The concentrations of HA and CS are in lg/g dry weight; percentage composition is given in parentheses. ...
... It is well established that GAGs are responsible for the water-storage capacity that gives articular cartilage elasticity. In normal bone, GAG's decrease because of age, therapy, or hormonal imbalance (17). On the other hand, elevated GAG levels have been shown to contribute to the worsening of the condition of patients suffering from autoimmune conditions such as rheumatoid arthritis and systemic lupus erythematosus The concentrations of HA and CS are in lg/g dry weight; percentage composition is given in parentheses. ...
Glycosaminoglycans and proteoglycans are macromolecules of the bone and are involved in the assembly, maturation, mineralization, and maintenance of the extracellular matrix. Heterotopic ossification is the rapid development of calcified bone tissue at ectopic sites of the body, mainly in soft tissues that normally do not ossify. The aim of this study was to characterize the molecular profiles of glycosaminoglycans and proteoglycans in normal and heterotopic bone samples to assess whether differences exist between orthotopic and heterotopic bone. Heterotopic bone tissues contained lower amounts of glycosaminoglycans compared to normal femoral bone. Structural analysis of chondroitin sulfate (CS) revealed that both heterotopic and normal femoral bones were composed mainly of 6-sulfated disaccharides. Quantitative differences in the disaccharide composition of CS, such as the decrease of 6-sulfated disaccharides in heterotopic bone with a concurrent increase of 4-sulfated and nonsulfated disaccharides, were found between ectopic bone and normal femoral bone. The proteoglycans decorin and aggrecan were both detected in all bone samples using specific antibodies. The detection of minor amounts of aggrecan in mature human bone, such as femoral bone, as well as in ectopic bone is described for the first time. These results may elucidate the phenomenon of ectopic bone formation and assist in early detection.
... GAGs, found primarily within the connective tissues including cartilage and heart valve leaflets, participate in numerous aspects of physiological and pathological function . These high molecular weight hydrophilic polysaccharides are responsible for the regulation of extracellular calcium as well as creating a hydrated milieu with nanomechanics conducive to absorbing stresses within articulating synovial joints12131415. Recent studies conducted by our group have illustrated the critical importance of GAGs in heart valve leaflet morphology, hydration and mechanical function [4]. Furthermore, studies have shown that GAGs are unremittingly lost from degenerated explanted BHVs as well as following in vitro accelerated fatigue testing of standard GLUT-fixed prosthetic tissue [5]. ...
Numerous crosslinking chemistries and methodologies have been investigated as alternative fixatives to glutaraldehyde (GLUT) for the stabilization of bioprosthetic heart valves (BHVs). Particular attention has been paid to valve leaflet collagen and elastin stability following fixation. However, the stability of glycosaminoglycans (GAGs), the primary component of the spongiosa layer of the BHV, has been largely overlooked despite recent evidence provided by our group illustrating their structural and functional importance. In the present study we investigate the ability of two different crosslinking chemistries: sodium metaperiodate (NaIO(4)) followed by GLUT (PG) and 1-Ethyl-3-(3 dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) followed by GLUT (ENG) to stabilize GAGs within BHV leaflets and compare resulting leaflet characteristics with that of GLUT-treated tissue. Incubation of fixed leaflets in GAG-degrading enzymes illustrated in vitro resistance of GAGs towards degradation in PG and ENG treated tissue while GLUT fixation alone was not effective in preventing GAG loss from BHV leaflets. Following subdermal implantation, significant amounts of GAGs were retained in leaflets in the ENG group in comparison to GLUT-treated tissue, although GAG loss was evident in all groups. Utilizing GAG-targeted fixation did not alter calcification potential of the leaflets while collagen stability was maintained at levels similar to that observed in conventional GLUT-treated tissue.
... It has been shown previously that GAGs play an important role in cartilage lubrication and biomechanical function [19]. Also it has been shown that GAGs are responsible for the water storage capacity that gives the articular cartilage its unique property of hydration and compressibility [20]. GAGs are present in the spongiosa layer of heart valve cusps and prevent local compressive buckling during valve function. ...
Bioprosthetic heart valves (BHVs) derived from glutaraldehyde crosslinked porcine aortic valves are frequently used in heart valve replacement surgeries. However, BHVs have limited durability and fail either due to degeneration or calcification. Glycosaminoglycans (GAGs), one of the integral components of heart valve cuspal tissue, are not stabilized by conventional glutaraldehyde crosslinking. Previously we have shown that valvular GAGs could be chemically fixed with GAG-targeted chemistry. However, chemically stabilized GAGs were only partially stable to enzymatic degradation. In the present study an enzyme inhibitor was incorporated in the cusps to effectively prevent enzymatic degradation. Thus, neomycin trisulfate, a known hyaluronidase inhibitor, was incorporated in cusps via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry followed by glutaraldehyde crosslinking (NEG). Controls included cusps crosslinked with either EDC/NHS followed by glutaraldehyde (ENG) or only with glutaraldehyde (GLUT). NEG group showed improved resistance to in vitro enzymatic degradation as compared to GLUT and ENG groups. All groups showed similar collagen stability, measured as a thermal denaturation temperature by differential scanning calorimetry (DSC). The cusps were implanted subdermally in rats to study in vivo degradation of GAGs. NEG group preserved significantly more GAGs than ENG and GLUT. NEG and ENG groups showed reduced calcification than GLUT.
One of the major strategies in tissue engineering is the biomimetic scaffold-based approach that aims at providing a near native like environment for cells to facilitate the regeneration of damaged/lost tissue. The extracellular matrix in native articular cartilage contains aligned collagen fibrils in the superficial (parallel to the articular surface) and deep zones (perpendicular to articular surface) of the tissue. Therefore, we hypothesized that scaffolds with aligned pore architecture may offer aligned collagen deposition upon cell seeding, and as a result, may enable enhanced chondrogenesis. We tested this hypothesis by comparing gelatin scaffolds with random and aligned pore architecture for their ability to differentiate infrapatellar fat pad derived mesenchymal stromal cells (IFP-MSCs) towards the chondrogenic lineage. The fabricated scaffolds with random and aligned pore architecture were comparable in terms of pore size, degree of crosslinking, equilibrium swelling ratio and in vitro degradation behaviour. However, scaffolds with aligned pore architecture demonstrated higher compressive modulus along with cellular infiltration and alignment in comparison to the scaffolds with random pore architecture. An in vitro chondrogenesis study of IFP-MSCs seeded in the developed scaffold systems revealed that scaffolds with aligned pore architecture supported better chondrogenesis in terms of sGAG and total collagen (histology and biochemical) and cartilage specific matrix deposition (immunofluorescence). Further, scaffolds with aligned pore architecture also supported oriented deposition of cell secreted collagen. Taken together, these results suggest that scaffolds with aligned pore architecture enhance in vitro chondrogenic differentiation of IFP-MSCs as compared to scaffolds with random pore architecture and hence could be a potential design criterion in the development of scaffolds for cartilage regeneration.
Estrogens have specific receptors spread out in various systems of the organism. The drastic hormonal fall after menopause may be followed by a series of effects, which may be more or less relevant in the different areas of the organism. The bone constitutes a field that clearly reflects that impact, and postmenopausal osteoporosis has received attention in one ad hoc chapter. The present chapter reviews the impact on functions of the central nervous system, particularly cognition and mood, the skeletal system, specifically osteoarthritis, and the cardiovascular system. The obvious interest of the chapter derives from the importance of the selected systems, which may house highly prevalent pathologies, with a considerable toll in morbidity, mortality, and dependence.
In this chapter, an overview is presented of the role of composite biomaterials as substitutes for soft tissue repair. In particular, those technological solutions where either biopolymers or synthetic biocompatible, biomimetic polymers have been used are highlighted. The potential of these biomaterials to fulfil clinical needs is underpinned by an overview of the composition and properties of the main extracellular matrix components. This overview allows the reader to critically assess the ability of the currently available composite biomaterials to perform in a clinical scenario. Examples of clinical applications are given in which composite biomaterials are fundamental to soft tissue aided repair. The clinical problems that arise when their use has been neglected, described.
Significantly elevated bone fluoride concentrations have previously been reported in a
population of eastern grey kangaroos (Macropus giganteus) resident near a fluoride-emitting
aluminum smelter in south-eastern Australia. This paper describes the skeletal and synovial joint
lesions observed post mortem in the same sample of kangaroos (n=76). The prevalence and severity of
skeletal lesions, specifically the formation of multiple, large, smooth exostoses over the diaphysis of
long bones (especially, but not exclusively, on the tibia, fibula and metatarsi), were positively
associated with bone fluoride concentration. So too were lesions of degenerative joint disease,
including periarticular osteophytosis, articular cartilage erosion/ulceration, synovial hyperplasia and
joint capsular fibrosis. Joint lesions were most commonly seen in the knee, hock and
metatarsophalangeal joints. This is the first paper to describe in detail the full range of lesions induced
by chronic fluorosis in a marsupial.
Osteoarthritis (OA) is a frequently occurring musculoskeletal disorder, leading to joint pain and disability. Although all tissues in the joint can be affected, the focus of this thesis is on changes in bone and cartilage. Evidence from literature suggests that estrogen may have an OA-protective effect. Both cartilage and bone are responsive to estrogen, and therefore the OA-protective effect of estrogen may act via both tissues. In this thesis, we aimed to obtain more insight in the protective effects of estrogen, by investigating the role of estrogen in OA at different levels:
The current literature concerning the effects of hormone depletion (by ovariectomy) and estrogen treatment on cartilage in animal models was reviewed in a systematic way.
Estrogen receptor knockout mice were studied to investigate the role of estrogen signalling in the osteoarthritic process.
Ovariectomy in mice was combined with an osteoarthritis trigger (or a saline injection) and changes in cartilage and bone of both proximal tibia and patella were described.
The effects of estrogen on cartilage without interference of surrounding tissues were investigated in a culture study of bovine cartilage explants.
In conclusion, the results in this thesis support the idea that OA is a multifactorial disease, in which the contribution of estrogen may be small. However the estrogen-induced changes, together with changes caused by other contributors, can result in osteoarthritis.
Information on the pathophysiology of glucocorticoid-induced osteoporosis (GIO) is limited, since its clinical picture often reflects a combined effect of glucocorticoids (GC) and the treated systemic disease (i.e., inflammation and immobility). In 50 healthy adult (30-mo-old) primiparous Göttingen minipigs, we studied the short-term (8 mo, n = 30) and long-term (15 mo, n = 10) effect of GC on bone and mineral metabolism longitudinally and cross-sectionally compared with a control group (n = 10). All animals on GC treatment received prednisolone orally at a dose of 1.0 mg x kg body wt(-1) x day(-1) for 8 wk and thereafter at 0.5 mg/kg body wt(-1) x day(-1). In the short term, GC reduced bone mineral density (BMD) at the lumbar spine by -47.5 +/- 5.1 mg/cm(3) from baseline (P < 0.001), which was greater (P < 0.05) than the loss [not significant (NS)] in the control group of -11.8 +/- 12.6 mg/cm(3). Calcium absorption decreased from baseline by -2,488 +/- 688 mg/7 days (P < 0.001) compared with -1,380 +/- 1,297 mg/7 days (NS) in the control group. Plasma bone alkaline phosphatase (BAP) decreased from baseline by -17.8 +/- 2.2 U/l (P < 0.000), which was significantly different (P < 0.05) from the value of the control group of -1.43 +/- 4.8 U/l. In the long term, the loss of BMD became more pronounced and bone mineral content (BMC), trabecular thickness, mechanical stability, calcium absorption, 25-hydroxyvitamin D(3), 1,25-dihydroxyvitamin D(3), and parathyroid hormone tended to be lower compared with the control group. There was a negative association between the cumulative dose of GC and BMD, which was associated with impaired osteoblastogenesis. In conclusion, the main outcomes after GC treatment are comparable to symptoms of GC-induced osteoporosis in human subjects. Thus the adult Göttingen miniature pig appears to be a valuable animal model for GC-induced osteoporosis.
The risk of regression after photorefractive keratectomy (PRK) and the tendency to develop keratectasia after laser-assisted in situ keratomileusis (LASIK) procedure is higher in women than men. Currently, interest is focused on the influence of oestrogen on corneal stability after corneal refractive surgery. The aim of this experimental study was to investigate the change in biomechanical properties of the cornea induced by oestrogen.
The influence of oestrogen was investigated in 12 fresh porcine corneas incubated in culture medium with 10 micromol/l beta-oestradiol for 7 days. A group of 12 porcine corneas incubated in culture medium without oestradiol for the same time served as a control group. Strips of cornea were cut and the stress-strain was measured in a biomaterial tester. The Young's modulus was calculated.
During incubation the thickness of the cornea changed in the control group by only 6.4% and in the oestradiol group by 12%. However, the difference in the biomechanical stress values at 10% strain was significantly larger. In the control group the stress value was 120.18+/-28.93 kPa and in the oestradiol group 76.87+/-34.63 kPa (p = 0.002), representing a reduction of the corneal stiffness by 36% due to the oestradiol treatment.
Oestrogen is a modulating factor of the biomechanical properties of the cornea that is not explainable only by an increased swelling. The significance of the hormone status of patients and its influence on the biomechanical stability of the cornea, a determining factor after refractive surgery, have been underestimated and may contribute to the development of keratectasia.
Glucocorticoids (GC) are used extensively in children and may cause growth retardation, which is in part due to the direct effects of GC on the growth plate. We characterised the ATDC5 chondrocyte cell line, which mimics the in vivo process of longitudinal bone growth, to examine the effects of dexamethasone (Dex) and prednisolone (Pred) during two key time points in the chondrocyte life cycle chondrogenesis and terminal differentiation. Additionally, we studied the potential for recovery following Dex exposure. During chondrogenesis, Dex and Pred exposure at 10 -8 M, 10 -7 M and 10 -6 M resulted in a significant mean reduction in cell number (28% vs 20%), cell proliferation (27% vs 24%) and proteoglycan synthesis (47% vs 43%) and increased alkaline phosphatase (ALP) activity (106% vs 62%), whereas the incidence of apoptosis was unaltered. Minimal effects were noted during terminal differentiation with both GC although all concentrations of Dex lowered apoptotic cell number. To assess catch-up growth the cells were incubated for a total of 14 days which included 1, 3, 7, 10 or 14 days exposure to 10 -6 M Dex, prior to the recovery period. Recovery of proteoglycan synthesis was irreversibly impaired following just one day exposure to Dex. Although cell number showed a similar pattern, significant impairment was only achieved following 14 days exposure. Irreversible changes in ALP activity were only noticed following 10 days exposure to Dex. In conclusion, GC have maximal effects during chondrogenesis; Dex is more potent than Pred and cells exposed to Dex recover but this may be restricted due to differential effects of GC on specific chondrocyte phenotypes.
Objective:
To determine if differences in biomechanical properties and biochemical composition exist between human patellar articular cartilage and the opposing femoral articular cartilage.
Design:
The biomechanical properties and biochemical composition of the articular cartilage of 17 knees from 13 donors were determined for four sites on the patella and three sites on the femur representing regions of contact at 30 degrees and 90 degrees of flexion. The material properties were determined by biphasic indentation testing, yielding the compressive aggregate modulus, HA, permeability, k, and Poisson's ratio, vs. The thickness of the cartilage at the indentation site, h, was also measured using a needle probe. Full-thickness samples of cartilage adjacent to each indentation site were used for wet weight, sulfated glycosaminoglycan content and hydroxyproline content determinations.
Results:
The patellar cartilage was found to have a lower compressive aggregate modulus by 30% (P < 0.001), higher permeability to fluid flow by 66% (P < 0.001) and greater thickness by 23% (P = 0.017) than that of the opposing femoral cartilage. The Poisson's ratios for both surfaces were found to be nearly zero. The water content of the patella was higher by 5% (P = 0.031) and the proteoglycan content lower by 19% (P = 0.030) than that of the femur. However, no differences were found between the collagen contents of the cartilages.
Conclusions:
Significant differences were found between the intrinsic material properties of the patellar cartilage and those of the femoral-trochlear cartilage. This variability of cartilage material properties with the patellofemoral joint may help explain why patellar cartilage has been frequently observed clinically to exhibit earlier and more severe fibrillation changes than the opposing femoral cartilage.
The type X collagen is a short chain collagen associated with calcific cartilage and/or the expression of the hypertrophic chondrocyte phenotype. In articular cartilage, type X collagen is restricted to the basal zone of calcified cartilage adjacent to the subchondral bone. However, during pathological change such as in osteoarthritis, the synthesis of type X collagen becomes more widespread but never extends to the articular surface. Using immunocytochemistry and fluorography of newly synthesised collagens, we report that surface articular chondrocytes (which occupy the uppermost 10-15% of the tissue depth) from normal human cartilage initiate de novo synthesis of both type X collagen and alkaline phosphatase when maintained in suspension culture.
Current efforts to monitor the mineral status of preterm infants fed human milk may not provide sufficient information on the distribution of body minerals. To investigate the body distribution of calcium and phosphorus during various degrees of mineral deficiency, neonatal miniature piglets were raised for 2 wk on diets differing only in calcium and phosphorus. Groups A, B, and C were fed 100%, 60%, and 20%, respectively, of the recommended amounts of calcium and phosphorus that, when adjusted for rates of growth, approximated the range of dietary intakes of preterm infants. Group C manifested biochemical and body-composition evidence of mineral deficiency when compared with group A: lower serum phosphorus; higher serum alkaline phosphatase activity; less fat-free tissue, calcium, and phosphorus in tibiae, vertebrae, and whole carcasses. Neonatal miniature piglets are useful for studying mineral deposition during mineral deficiency in preterm infants.
To characterize chondrocyte alkaline phosphatase (ALP) distribution and expression in cartilage and monolayer cultures.
Sections of bovine articular cartilage or chondrocyte monolayer cultures were stained for ALP activity. Surface ALP was released with bacterial phosphatidylinositol specific phospholipase C (PI-PLC). The levels of ALP mRNA were determined by Northern blot analysis using a cDNA probe to bovine ALP.
Chondrocyte ALP activity dissolves calcium pyrophosphate dihydrate (CPPD) crystals. About 5% of the total chondrocytes contained ALP activity. The ALP positive cells were present only in the deep layers of articular cartilage adjacent to the subchondral bone. Thirty to forty percent of the total ALP activity was present on the chondrocyte surface and was released by PI-PLC. Both chondrocyte ALP activity and mRNA levels decreased with time in culture. However, continuous dexamethasone treatment stimulated the expression of ALP in ALP positive chondrocytes, sufficiently to replace all of the chondrocyte surface ALP released following PI-PLC treatment.
Since ALP hydrolyzes pyrophosphate and dissolves CPPD crystals, our data suggest that regulation of chondrocyte ALP activity and expression in cartilage may prove useful for the development of a specific therapy CPPD arthropathy.
To investigate the suitability of the pig as animal model for postmenopausal osteoprosis, effects of ovariectomy (OVX) on bone metabolism and histology were studied in two groups of sows (9 months, nulliparous or 35 months, multiparous). A standard diet of about 1.5 % calcium (Ca) was fed till sacrifice at either 12 or 20 months post OVX when mineral content and histology were studied in representative bone specimens of proximal tibia, iliac crest and lumbar vertebrae. At 4, 8, 12, and 18 months post OVX, total and bone-specific alkaline phosphatase (APt, APb) calcidiol, calcitriol and parathyroid hormone (PTH) were measured in plasma.
In young sows OVX did not significantly affect plasma variables except for calcitriol, which was higher at 4 months post OVX. No significant differences between OVX or control animals were observed in the variables of bone chemical and histological analyses, neither 12 nor 20 months post OVX.
In multiparous sows OVX significantly increased PTH plasma concentrations at 8 months post OVX and plasma calcitriol, APt and APb at 12 months post OVX. All effects were moderate and transient. OVX did not significantly affect the variables of bone chemical and histological analyses neither 12 nor 20 months post OVX.
Although undoubtedly the clinical-chemical changes observed were not accompanied by any histomorphometric signs of osteopenia/osteoporosis, it must be left to future experiments as to whether this resulted from the ample calcium supply provided. This possibility is supported by recent observations showing that porcine osteopenia could be induced by OVX in animals maintained on only 0.75 % dietary calcium but not on higher (0.9 %) Ca regimens (33).
Osteoarthritis (OA) is increasingly prevalent in the years after menopause. Epidemiological data suggest that the use of oestrogen replacement therapy (ERT) may protect against knee OA.
To test the hypothesis that long term ERT (longer than five years) is associated with increased knee cartilage in postmenopausal women.
The study involved 81 women (42 current users (> or = five years) of ERT and 39 who had never used it). Articular cartilage volumes were determined by processing images acquired in the sagittal plane using a T1 weighted fat suppressed magnetic resonance sequence on an independent work station.
After bone size had been accounted for, ERT users had higher tibial cartilage volume than non-users. Total tibial cartilage volume was 7.7% (0.23 ml) greater in the group of ERT users (2.98 (0.47) ml; mean (SD)) than in the untreated group (2.75 (0.50) ml). The difference, after adjustment for the significant explanatory factors (years since menopause, body mass index, age at menopause, and smoking), between the ERT users and non-users increased from 0.23 ml to 0.30 ml (95% confidence interval 0.08 to 0.52, p=0.008). These differences persisted after exclusion of women with OA.
After adjustment for multiple confounders, women using long term ERT have more knee cartilage than controls. This may indicate that ERT prevents loss of knee articular cartilage.
Experimental evidence suggests that recommended dosages of some corticosteroids used clinically as antiinflammatory agents for treating arthropathies damage articular cartilage, but low dosages may be chondroprotective. The purpose of this study was to evaluate how different concentrations of methylprednisolone affect chondrocyte function and viability. Articular cartilage and chondrocytes were obtained from young adult horses, 1.5–3.5 years of age. Corticosteroid‐induced changes in collagen expression were studied at the transcriptional level by Northern blot analyses and at the translational level by measuring [³H]‐proline incorporation into [³H]‐hydroxyproline. Fibronectin mRNA splicing patterns were evaluated with ribonuclease protection assays. Cytotoxicity was studied using erythrosin B dye exclusion. Steady‐state levels of type II procollagen mRNA decreased without concurrent changes in type I procollagen expression as the medium methylprednisolone concentrations were increased from 1 × 10¹ to 1 × 10⁸ pg/ml, dropping below 10% of control values by 1 × 10⁵ pg/ml. Cytotoxicity occurred as methylprednisolone levels were increased further from 1 × 10⁸ to 1 × 10⁹ pg/ml. Changes in total collagen (protein) synthesis were less pronounced, but also demonstrated significant suppression between 1 × 10⁴ and 1 × 10⁸ pg/ml. Corticosteroid‐induced changes in fibronectin isoform levels were evaluated in articular cartilage samples without in vitro culture. The cartilage‐specific (V + C)⁻ isoform was suppressed in both normal and inflamed joints by a single intraarticular injection (0.1 mg/kg) of methylprednisolone. Combined, these data indicate that methylprednisolone suppresses matrix protein markers of chondrocytic differentiation. Decreased and altered chondrocyte expression of matrix proteins likely contributes to the pathogenesis of corticosteroid‐induced cartilage degeneration. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
The ultrastructure changes in the articular cartilage of rabbits, subjected to daily treatment with hydrocortisone, 10 mg/kg body weight, i.m. over a period of 6–36 days, and of mice following adrenal extirpation are investigated. The changes of the cellular organelles established in both experimental groups demonstrate the presence of an inhibitory effect of hydrocortisone equally on protein and carbohydrate synthesis.
Estradiol valerate did not ameliorate experimentally induced osteoarthritis in the rabbit. Both normal and osteoarthritic cartilage were susceptible to estradiol suppression of proteoglycan synthesis. Proteoglycan concentration was not diminished with estradiol, suggesting estradiol suppression of proteoglycan catabolism. The severity of osteoarthritis was unchanged despite markedly decreased proteoglycan synthesis in the estrogen treated animals. Osteophyte proteoglycan metabolism differed from other osteoarthritic lesions. Differences in the metabolism of femoral and tibial articular cartilage were observed.
Die Therapie der Osteoporosen richtet sich nach einfachen Grundsätzen. Liegt eine Osteoporose mit hohem Knochenumsatz vor, sollte dieser Umsatz zunächst gebremst werden. Hierzu dienen Bisphosphonate, das Raloxifen und in seltenen Fällen ein Calcitonin. Ist der hohe Knochenumsatz gebremst oder liegt von Anfang an eine Osteoporose mit niedrigem Knochenumsatz vor, sollte ein knochenanaboles Kombinationsschema begonnen werden. Dies besteht aus der Induktion des Kollagenes vom Typ I durch Parathormon oder Fluoride. Das so synthetisierte Osteoid sollte durch Kalzium, Vitamin D oder dessen Metabolite mineralisiert werden. Zusätzlich sollte der Knochenabbau in der Perimenopause durch Östrogene und Gestagene, bei einer Patientin mit einem rezeptorpositiven Mammakarzinom durch Antiöstrogene und nach dem Klimakterium durch Raloxifen oder Bisphosphonate gebremst werden.
Im höheren Alter liegt häufig keine reine Osteoporose, sondern eine durch mangelnde Vitamin-D-Versorgung bedingte Osteoporomalazie mit untermineralisiertem Osteoid vor. In einer solchen Situation genügt häufig die Gabe von Vitamin D und Kalzium, um eine ausreichende Mineralisation zu erreichen. Gleichzeitig muss eine muskelaufbauende, physikalische Therapie erfolgen, da sich der Knochen an die jeweils vorhandene Muskelmasse adaptiert.
Morphometrical and histochemical techniques were used to demonstrate changes to the cartilage layer of the rat temporomandibular joint condyle following chronic exposure to fluoride. An increase in thickness of the cartilage layer was noted in rats given 100 parts per million sodium fluoride in drinking water. No significant changes were observed with either control or low dose (10 parts per million) groups. The observed thickening was attributable to an increase in number and size of cells of the lower hypertrophic zone. Accumulations of glycogen were observed in these cells, which reflects the inhibitory effect of fluoride on glycolysis. Stimulation of chondrocytes by fluoride may have delayed the normal processes of capillary invasion, resulting in thickening of the cartilage layer. No changes to staining patterns of immature or mature types of collagen were observed, nor did the staining pattern of detectable glycosaminoglycans change due to fluoride.
The present study aimed to describe the ultrastructural localization of alkaline phosphatase (AP) activity in articular-epiphyseal growth cartilage of the commercial pig and the minipig of wild hog ancestry, comparing areas with a normal endochondral ossification with those where the calcification of the matrix is insufficient, as in osteochondrotic cartilage. Intense AP activity was primarily present in the cytoplasm, the plasmalemmae, the long cellular processes and the matrix vesicles budding off from proliferative and hypertrophic chondrocytes in those areas of cartilage where normal calcification appeared. In the osteochondrotic cartilage, the only detectable AP activity was restricted to a few morphologically viable hypertrophic cells in the surroundings of the lesion. The lack of AP activity could partially explain the insufficient calcification of the osteochondrotic cartilage.
Articular cartilage varies in ultrastructure and composition with distance from the articular surface. We have cultured chondrocytes from different zones of pig articular cartilage and investigated whether there are intrinsic differences in their behaviour that might account for the variation observed in intact tissue. On isolation, cells from the upper third of the cartilage were smaller than those of the lower third, but this difference was not maintained in culture. Upper zone cells attached and spread more slowly than lower zone cells; morphological differences between the two populations could be seen for several weeks. The growth rates of the two populations were similar, but upper zone cells reached a lower confluent density. Levels of protein synthesis were similar for both populations, but upper zone cells deposited less proteoglycan in the cell layer. On isolation, the percentage of upper zone cells that stained positive with MZ15, a monoclonal antibody to keratan sulphate, was smaller than the percentage of lower zone cells, but this difference was lost after several days in culture. Nevertheless, the keratan sulphate content of proteoglycan synthesised by lower zone chondrocytes at high density was greater than of that synthesised by upper zone cells. The proportion of nonaggregating proteoglycan was greater in upper than lower zone cartilage and this difference was also observed in long-term cultures. proteoglycans were further characterised by composite and polyacrylamide gel electrophoresis and by immunoblotting; differences detected in cartilage extracts were not, however, maintained in culture; instead, the small proteoglycans synthesised by both upper and lower zone cells varied with plating density. Finally, alkaline phosphatase, a marker of hypertrophic, calcifying cartilage, was only expressed in lower zone cultures. We conclude that some of the observed heterogeneity of articular cartilage reflects intrinsic differences between the cells of different zones, whereas some may reflect the response of chondrocytes to different environmental conditions.
The purpose of the experiment was to assess the effects of fluoride (F-) on the remodeling process of cortical bone. Sixteen pigs, eight experimental animals receiving a supplement of 2 mg F-/kg b.w. and eight controls, were studied in individual sites from age 8 to 14 months. At slaughter samples of cortical bone were obtained from the right femur and embedded undecalcified. A new stereologic model based on fluorochrome tissue time markers and isotropic uniform random histologic sections was implied in order to obtain information in three-dimensional terms about dynamic aspects of remodeling. The rate of remodeling was increased in cortical bone from pigs receiving F- due to an increased activation of new remodeling. A doubling of the length density of resorptive and formative osteons was observed, although the change was statistically significant for the formative osteons only. An 11% decrease in depth of resorption and an 8% decrease in thickness of new bone formed led to a small decrease in the radius of Haversian canals in the fluorotic bone. The porosity of cortical bone was slightly but significantly increased. At formative sites the osteoid thickness and the mineralization rate were not significantly changed by F-. It was concluded that the observed changes cannot be explained by F- induced changes in a single cell. Fluoride appears to affect all cells involved in remodeling by direct or indirect mechanisms.
The dimethylmethylene blue assay for sulphated glycosaminoglycans has found wide acceptance as a quick and simple method of measuring the sulphated glycosaminoglycan content of tissues and fluids. The available assay methods have lacked specificity for sulphated glycosaminoglycans in the presence of other polyanions, however, and have not discriminated between the different sulphated glycosaminoglycans. We now describe a modified form of the dimethylmethylene blue assay that has improved specificity for sulphated glycosaminoglycans, and we show that in conjunction with specific polysaccharidases, the dimethylmethylene blue assay can be used to quantitate individual sulphated glycosaminoglycans.
The distribution of alkaline phosphatase activity in human articular cartilage from normal and osteoarthritic joints has been examined by an electron microscope technique, probably for the first time. In osteoarthritic cartilage chondrocytes and matrix vesicles close to the tidemark were positive for alkaline phosphatase activity. Large numbers of matrix vesicles were found within the extracellular matrix of osteoarthritic cartilage, and there is a specific relation between phosphatase activity, matrix vesicles, and initial mineral formation in the tidemark region of articular cartilage.
(1) Pharmacological concentrations (greater than 10(-5) M) of 17 beta-oestradiol inhibited 35S-labelled proteoglycan synthesis in bovine articular cartilage explant cultures. They also inhibited 35S-labelled proteoglycan synthesis and 3H-labelled protein synthesis in cell cultures of chondrocytes from bovine articular cartilage and Swarm rat chondrosarcoma. Maximal inhibition was about 30-50%. Physiological concentrations (10(-9)-10(-8) M) of oestradiol had no effect on the synthesis of either protein or proteoglycan. (2) The inhibitory action of high concentrations of oestradiol on these biosynthetic pathways is not common to all steroids since 10(-4) M cortisol had no effect on articular chondrocyte cell cultures. 10(-4) M testosterone had a similar action to oestradiol. (3) Neither physiological nor pharmacological concentrations of 17 beta-oestradiol had any effect on 35S-labelled proteoglycan turnover in the cartilage explant system. (4) 10(-5) M oestradiol inhibited cell division in cultures of articular chondrocytes which had entered the log growth phase. 10(-7) M oestradiol had no effect on articular chondrocyte growth. (5) In male rats implanted with silastic capsules releasing 17 beta-oestradiol, increase in body weight was retarded by about 25% over a period of 6 weeks, compared to control rats. Rat chondrosarcoma grew to the same size in oestrogen-treated rats as it did in controls. (6) Oestrogen receptors could not be detected in freshly isolated bovine articular chondrocytes or in rat chondrosarcoma. (7) In conclusion, neither the mitotic rate of articular chondrocytes nor their proteoglycan metabolism is under the direct physiological control of oestradiol. Growth and biosynthetic activity of the rat chondrosarcoma chondrocytes are independent of either direct control by the hormone or control effected by oestradiol regulation of a second hormone or growth factor.
Subchondral bone and calcified cartilage from a femoral head of a 74-year-old osteoporotic woman treated for 30 months with sodium fluoride were analyzed. The fluoride content of the calcified tissues was determined by a specific ion electrode, and the topographic distribution pattern of fluoride was determined with an electron microprobe. The fluoride content in calcified cartilage (0.39% of ash) was higher than in neighboring subchondral bone (0.28% of ash). Line scan and X-ray images indicated a high concentration of fluoride in the outer layer of calcified cartilage lining the uncalcified cartilage, as well as in the inner layer of the subcortical endosteal bone. This study shows that calcified cartilage is an important site of fluoride deposition, and suggests that the accumulation of fluoride is related to the calcification process.
The application of the “critical electrolyte concentration” (CEC) concept to the differentiation of acidic glycosaminglycans (mucopolysaccharides) is described. Alcian Blue 8GX stains with increasing selectivity as increasing amounts of magnesium chloride are incorporated into the dye solution. Model experiments with pure polyanions, or artifically carboxylated, phosphorylated and sulphated liver sections, showed that binding of dye to carboxylate or phosphate groups ceased at low electrolyte concentrations (< 0.3M) whereas dye continued to be held by sulphate ester groups at concentrations five to ten times as high. The similarity to the well established cetylpyridinium system for polyanion fractionation is discussed.
Tidemark is an interface which may better be defined by biochemical methods than by morphology. It originates, by chondrocyte activity, between calcified and noncalcified cartilage layers of any kind, hyaline or fibrous, in areas exposed to either loading (joint) or pulling (insertion). In the articular cartilage it appears with skeletal maturation, in other localizations it is age-independent. It should be regarded as a special instance of a broader phenomenon of the calcification/mineralization front. Inside the joint cartilage its changes reflect the slow remodelling of the calcified layer and its inapparent shift towards the surface of the articular cartilage. In the marginal transitional zone of the joint, tidemark smoothly passes into the periosteum. Chondrocytes on both sides of the tidemark are positive for alkaline phosphatase and the positive reaction continuously goes on to the periosteum.
Uptake of [35S]sulfate by segments of rat costal cartilage during culture was greatly stimulated when freshly prepared phenylmethylsulfonyl fluoride or diisopropylfluorophosphate was included in the incubation medium. By contrast, hydrolysed diisopropylfluorophosphate, sodium fluoride or soybean trypsin inhibitor did not stimulate [35S]sulfate uptake. Incorporation of four other radioactive precursors of cartilage synthesis was almost completely suppressed during cartilage incubation in the presence of phenylmethylsulfonyl fluoride. However, stimulation of [35S]sulfate binding by the latter was shown to occur at sites other than on glycosaminoglycan molecules and to a similar degree with both active and inactivated cartilage. These and other data indicate that the stimulatory effect of phenylmethylsulfonyl fluoride on [35S]sulfate uptake is independent of normal metabolic processes, and may involve the binding of phenylmethylsulfonyl fluoride to cartilage proteins.
Therapy with low-dose corticosteroids is commonly used to treat allergic and autoimmune diseases. Long-term use of corticosteroids can lead to loss of bone mineral density and higher risk for vertebral fractures. Calcium and vitamin D3 supplementation is rational therapy for minimizing bone loss, but little evidence for its effectiveness exists.
To assess 1) the effects of supplemental calcium and vitamin D3 on bone mineral density of patients with rheumatoid arthritis and 2) the relation between the effects of this supplementation and corticosteroid use.
2-year randomized, double-blind, placebo-controlled trial.
University outpatient-care facility.
96 patients with rheumatoid arthritis, 65 of whom were receiving treatment with corticosteroids (mean dosage, 5.6 mg/d).
Calcium carbonate (1000 mg/d) and vitamin D3 (500 IU/d) or placebo.
Bone mineral densities of the lumbar spine and femur were determined annually.
Patients receiving prednisone therapy who were given placebo lost bone mineral density in the lumbar spine and trochanter at a rate of 2.0% and 0.9% per year, respectively. Patients receiving prednisone therapy who were given calcium and vitamin D3 gained bone mineral density in the lumbar spine and trochanter at a rate of 0.72% (P = 0.005) and 0.85% (P = 0.024) per year, respectively. In patients receiving prednisone therapy, bone mineral densities of the femoral neck and the Ward triangle did not increase significantly with calcium and vitamin D3. Calcium and vitamin D3 did not improve bone mineral density at any site in patients who were not receiving corticosteroids.
Calcium and vitamin D3 prevented loss of bone mineral density in the lumbar spine and trochanter in patients with rheumatoid arthritis who were treated with low-dose corticosteroids.
Early degeneration of cartilage is accompanied by a loss of proteoglycans and consequent changes in the content of water. Conventional magnetic resonance imaging (MRI) cannot reliably detect this change, since the relaxation properties of the cartilage are dominated by its collagen content. The applicability of a positively charged nitroxide as an MRI contrast agent in detection of the content of the negatively charged proteoglycans within the cartilage was investigated. The results from both MRI and electron paramagnetic resonance (EPR) spectroscopy indicate that the accumulation of the contrast agent reflects the amount of proteoglycans within the cartilage, presumably due to the electrostatic interactions between the negatively charged proteoglycans and the positively charged nitroxide. Such a contrast agent could be useful in the detection and study of early stages of the degeneration of joints.
The zone of calcified cartilage (ZCC) forms an important interface between cartilage and bone for transmitting force, attaching cartilage to bone, and limiting diffusion from bone to the deeper layers of cartilage. The height of the ZCC is a relatively constant percent of articular cartilage and the height is maintained by a balance between progression of the tidemark into the unmineralized cartilage and changing into bone by vascular invasion and bony remodeling. During its formation, the cells that form the ZCC have properties similar to the cells of the growth plate. In the adult, the ZCC becomes quiescent but not inactive. The ZCC may be reactivated in osteoarthritis and may progressively calcify the unmineralized cartilage. This might contribute to cartilage thinning which would increase the concentration of forces across the uncalcified cartilage leading to more damage. Although the subchondral bony plate remodels extensively in osteoarthritis, there is little evidence that a change in the biomechanics of the plate directly initiates the osteoarthritic process in cartilage. However, increased repair by endochondral ossification of vertical cracks in the ZCC that penetrate into the marrow space could contribute to progression via changes in the ZCC.
Das Göttinger Minipig eignet sich aufgrund der Ähnlichkeit bezüglich seiner Labordaten und Knochenheilungsraten sehr gut als Versuchstier und wird in jüngster Zeit immer häufiger beschrieben (1, 2, 3, 8, 14, 16, 17, 19).
Da bisher bei Miniaturschweinen noch keine Hüftprothesen implantiert wurden bestand die Frage: Ist die TEP-Implantation technisch möglich? Anhand von 3 Minipigkadavern aus anderen Tierversuchen wurde der Zugangsweg erarbeitet, und die Meßdaten für die Prothesen und Werkzeuge erstellt. Anhand von 4 Vorversuchsoperationen wurden ein Prothesendesign und ein Operationsverfahren entwickelt und standardisiert. Danach wurden 10 erwachsene Tiere operiert und 1 Jahr beobachtet.
Man sollte bei der Auswahl der Tiere darauf achten, daß das Gewicht zwischen 30 kg und 45 kg liegt, da sonst der Schaft zu klein ist. Die Tiere sind während der Narkose gut zu steuern, und der Blutverlust ist äußerst gering. Eine Entlüftung des Markraumes ist beim zementieren nicht notwendig, da es beim Minipig in keinem der Fälle zu einer Kreislaufdepression kam. Bewährt hat sich ein anterolateraler Zugang bei Seitenlage des Tieres. Operationstechnisch hat sich bewährt: 1. Der Hautschnitt muß etwa eine handbreit cranial vom Schwanz des Tieres leicht bogenförmig gezogen werden. 2. Die Schenkelhalsosteotomie unter maximaler Außenrotation erfolgt V-förmig. 3. Mit einer 23,5 mm Fräse wird das Acetabulum aufgefräst. 4. Der Schaft sollte beim Minipig zuerst implantiert werden. Alle 10 Tiere laufen bei primärer Wundheilung ohne Schwierigkeiten. Bei der Röntgenkontrolle nach einem halben und einem Jahr fand sich nur bei einem der Tiere eine radiologische Pfannenlockerung.
Bisher hat sich das GMS als einfaches Tiermodell in der Hüftendoprothetik bewährt.
To clarify the relation between changes in bone density, the treated disease, and dose of corticosteroids prescribed.
MEDLINE database (1966-95) and bibliographic searches selected cohorts of patients with rheumatoid arthritis (RA) and non-RA patients, studied by reliable serial bone density measurements.
Two randomized controlled trials in early RA found greater lumbar bone loss after corticosteroid treatment (pooled effect size at 6 mo 3.9%; 95% CI: 1.9, 6.0%). The other studies included 66 patients with RA taking mean 7 mg prednisone/day; 371 "untreated" RA patients; and 216 non-RA patients taking mean 20 mg prednisone/day. Lumbar bone mass changed (weighted mean) 0.0% (-0.6, 0.7%) per year in steroid treated RA, -0.6% (-0.9, -0.2%) in untreated RA, and -4.7% (-5.2, -4.3%) in non-RA. Femoral neck changed -3.0% (-4.2, -1.8%), -0.7% (-1.0, -0.3%), and -1.5% (-2.5, -0.4%), respectively. In RA, most bone was lost in the first half year, and in early or uncontrolled disease.
In patients with RA bone loss is limited, influenced by the interaction of disease characteristics and low dose corticosteroid therapy. In contrast, non-RA patients taking higher doses of corticosteriods may loss clinically relevant amounts of bone (i.e., > 5%) within one year.
Bovine cartilage explants were treated with 100 ng/ml recombinant human interleukin-1beta (IL-1beta) or 1 microM all-trans retinoic acid (RA) and changes in biochemical, biomechanical, and physicochemical properties were assessed. Additionally, samples cultured with IL-1beta or RA were treated with 4 microM recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) or a synthetic metalloproteinase inhibitor (L-758,354) to inhibit this degradation. Treatment with IL-1beta or RA each resulted in >90% GAG loss after 8 days in culture. Addition of TIMP or L-758,354 to the culture media inhibited IL-1beta-induced loss of tissue GAG by 40 and 65%, respectively, and inhibited RA-induced GAG loss by 35 and 65%, respectively. Analysis of degradation products in the culture media using a G1 antibody indicated that IL-1beta- and RA-treated plugs released 68-kDa fragments of aggrecan, corresponding to a segment of the aggrecan core protein from the G1 domain to the C-terminus NITEGE, consistent with "aggrecanase" activity. Release of the G1 fragment was inhibited by treatment with L-758,354. Both IL-1beta and RA induced significant loss of hyaluronan from cartilage explants after 8 days of exposure and HA loss was also inhibited by addition of L-756,354 to the culture media. IL-1beta, but not RA, induced a significant increase in swelling ratio (wet weight in 0.01 M NaCl normalized to wet weight in DMEM) after 8 days in culture, consistent with degradation of the collagen network, and the increase in tissue swelling was inhibited by treatment with TIMP-1 or L-758,354. Exposure to IL-1beta or RA resulted in significant changes in cartilage physical properties including streaming potential, equilibrium modulus, hydraulic permeability, and electrokinetic coupling coefficient after 8 days in culture, and these changes were inhibited by 40-90% by exposure to TIMP and by 50-90% by exposure to L-758,354. Measurement of dynamic streaming potential showed that changes due to treatment with IL-1beta alone were highly dependent in compression frequency, with dramatic changes seen at high frequency prior to changes in mechanical properties, and little initial change seen at low frequency. Streaming potential and equilibrium modulus of explants treated with RA decreased to 10% of their initial values after 8 days in culture, but decreased to only 40 and 90%, respectively, when treated with RA plus TIMP-1.
Articular cartilage covering the bone ends at the joint shows different chemical composition in different regions, depending on the mechanical and biological properties of that region. Several studies have shown a relationship between the chemical composition of the cartilage and biomechanical forces. In the present study we analysed five different knee joints divided into the following regions: F1-medial and lateral border of the patellar surface, F2-patellar surface of the femur, F3-medial and lateral condyles, P-articular surface of the patella and T-medial and lateral condyle of the tibia. The main glycosaminoglycan (GAG) present in these regions was chondroitin sulfate. Analysis of total GAG after digestion of the tissue with papain showed that in F2 and F3 there was a larger quantity of GAG/mg tissue, probably due to the dynamic character of the biomechanical forces in these regions. No significant differences were found for the extract and D1 fractions of the different regions. Analysis of the D4 fraction showed that the protein content was higher in the F3 and P regions than in their opposite T and F2 regions. The differences among the five regions may be a result of the non-uniform presence of biomechanical forces supported by these regions. It is important to consider that the intensity and direction of stress in different parts of a tissue may influence the composition of the extracellular matrix.
The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2 mM sodium fluoride, whereas for osteoclasts 50-100 mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralizing zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.
Bisphosphonates have emerged as a valuable treatment for postmenopausal osteoporosis. Bisphosphonate treatment is usually accompanied by a 3-6% gain in bone mineral density (BMD) during the first year of treatment and by a decrease in bone turnover. Despite low bone turnover, BMD continues to increase slowly beyond the first year of treatment. There is evidence that bisphosphonates not only increase bone volume but also enhance secondary mineralization. The present study was conducted to address this issue and to compare the effects of continuous and intermittent bisphosphonate therapy on static and dynamic parameters of bone structure, formation, and resorption and on mineral properties of bone. Sixty dogs were ovariohysterectomized (OHX) and 10 animals were sham-operated (Sham). Four months after surgery, OHX dogs were divided in six groups (n = 10 each). They received for 1 year ibandronate daily (5 out of 7 days) at a dose of 0, 0.8, 1.2, 4.1, and 14 microg/kg/day or intermittently (65 microg/kg/day, 2 weeks on, 11 weeks off). Sham dogs received vehicle daily. At month 4, there was a significant decrease in bone volume in OHX animals (p < 0.05). Doses of ibandronate >/= 4.1 microg/kg/day stopped or completely reversed bone loss. Bone turnover (activation frequency) was significantly depressed in OHX dogs given ibandronate at the dose of 14 microg/kg/day. This was accompanied by significantly higher crystal size, a higher mineral-to-matrix ratio, and a more uniformly mineralized bone matrix than in control dogs. This finding lends support to the hypothesis that an increase in secondary mineralization plays a role in gain in BMD associated with bisphosphonate treatment. Moreover, intermittent and continuous therapies had a similar effect on bone volume. However, intermittent therapy was more sparing on bone turnover and bone mineral properties. Intermittent therapy could therefore represent an attractive alternative approach to continuous therapy.
The enzymatic processes underlying the degradation of aggrecan in cartilage and the corresponding changes in the biomechanical properties of the tissue are an important part of the pathophysiology of osteoarthritis. Recent studies have demonstrated that the hexosamines glucosamine (GlcN) and mannosamine (ManN) can inhibit aggrecanase-mediated cleavage of aggrecan in IL-1-treated cartilage cultures. The term aggrecanase describes two or more members of the ADAMTS family of metalloproteinases whose glutamyl endopeptidase activity is known to be responsible for much of the aggrecan degradation seen in human arthritides. In this study we examined the effect of ManN and GlcN on aggrecanase-mediated degradation of aggrecan induced by IL-1alpha and the corresponding tissue mechanical properties in newborn bovine articular cartilage. After 6 days of culture in 10 ng/ml IL-1 plus ManN, mechanical testing of explants in confined compression demonstrated that ManN inhibited the IL-1alpha-induced degradation in tissue equilibrium modulus, dynamic stiffness, streaming potential, and hydraulic permeability, in a dose-dependent fashion, with peak inhibition ( approximately 75-100% inhibition) reached by a concentration of 1.35 mM. Aggrecan from explants cultured in IL-1 was found by Western analysis to be almost entirely processed down to the G1-NITEGE(373) end product. Addition of ManN or GlcN was found to produce 75-90% inhibition of this cleavage, but the proportion of aggrecan remaining in the tissue which was cleaved at aggrecanase sites in the chondroitin sulfate (CS)-rich region (Glu(1501) and Glu(1687)) was higher than with IL-1 alone. This result suggests that the preservation of mechanical properties by hexosamines in explants is primarily due to inhibition of cleavage at the Glu(373) site in the interglobular domain. While the precise mechanism by which hexosamines function in this system is unclear, the present analysis suggests that the mechanical properties examined may be predominantly a function of electrostatic repulsion due to the charged CS chains in the tightly packed repetitive sequences of the CS-1 region.
It has been suggested that hormone replacement therapy (HRT) may protect against osteoarthritis (OA). The aim of this paper was to assess the association between HRT and radiographically defined patterns of OA.
475 consecutive women aged 50 years or older (mean age 66.1) who underwent hip or knee joint replacement because of advanced OA in four hospitals in south west Germany were enrolled in a cross sectional study. Participants underwent a standardised interview including detailed history of medication use and a physical examination. Furthermore, radiographs of the joint being replaced and of the contralateral joint as well as of both hands were obtained. Patients were categorised as having bilateral or unilateral OA according to the presence or absence of radiographic OA in the contralateral joint. If radiographic OA of different hand and finger joint groups was present, participants were categorised as having generalised OA (GOA). Logistic regression was used to estimate odds ratios and their 95% confidence intervals for the association between HRT and bilateral or GOA while adjusting for potential confounders.
Fifty five women (11.6%) were using HRT. The median duration of use was 5.4 years. The prevalence of bilateral and GOA was similar among users of ORT (86.3% and 27.5%, respectively) and among non-users of HRT (88.7% and 35.7%, respectively). After adjustment for potential confounding factors, the odds ratios (95% confidence intervals) of bilateral OA and GOA among HRT users compared with non-users was 1.21 (0.48, 3.03) and 1. 21 (0.53, 2.74), respectively.
Despite limited generalisability because of the selective study sample, these data do not support the hypothesis that HRT acts as a systemic protective factor against OA.
Numerous reports of negative effects as well as protective effects of glucocorticoids on articular cartilage convinced us to study the influence of hydrocortisone on aggrecan synthesis of isolated phenotypically stable human articular chondrocytes cultured in two different matrices.
Macroscopically normal human articular cartilage was obtained from femoral condyles within 24 hours postmortem. Chondrocytes were isolated and cultured in gelled agarose or in alginate. After 14 days in culture, hydrocortisone was added for 5 days at concentrations ranging from 0.005 microgram to 1 mg/ml for the agarose cultures and from 0.005 microgram to 1 microgram/ml for the alginate culture system. Aggrecan synthesis was measured by the incorporation of 35Sulphate, and the proportion of neosynthesized aggrecan that bound to hyaluronan to form aggrecan aggregates was analyzed by gel chromatography.
At concentrations from 0.005 to 1 microgram/ml, hydrocortisone was found to produce a similar dose-dependent stimulation of aggrecan synthesis in both matrices. The synthesis of aggrecans remained at the same level for concentrations of 1 microgram/ml up to 100 micrograms/ml of hydrocortisone. When supraphysiological concentrations of hydrocortisone were added the aggrecan synthesis rate plateau declined. Simultaneously with the increase in aggrecan synthesis, the proportion of low-molecular weight 35S-proteoglycans decreased in favour of 35S-aggrecan aggregates and monomers in the agarose system. The chondrocytes cultured in alginate showed this increase of aggrecan aggregates and monomeric aggrecans in both the cell-associated and the inter-territorial matrix.
Hydrocortisone is a stimulator of aggrecan synthesis by normal human articular chondrocytes cultured in vitro. The two culture systems (agarose and alginate) tested in this experiment showed a comparable aggrecan synthesis rate, increasing under the influence of hydrocortisone at concentrations up to 1 microgram/ml. The proportions of 35Sulphate incorporated in aggrecan aggregates and monomeric aggrecan were also higher under the influence of hydrocortisone.
The content of different forms of tissue water was studied in the normal articular cartilage and osteoarthrosis cartilage and its structural components: collagen, potassium hyaluronate, sodium chondroitinsulphate and its complexes. In the components of cartilage matrix a few of fractions of bound water different in the strength of binding are present. At the maximal humidity, all water in collagen binds with the active groups of biopolymers and in the glycosaminoglycans, in addition to bound water, are present, two crystal forms of freezing water (free water) at least. The quantity of free water in the collagen-chondroitin sulphat membrane, is increased with the increase of chondroitin sulphate. In the collagen-hyaluronate complex, fraction of free water is found only at the low concentration of hyaluronate kalium. It was shown that in the hyalin cartilage, in different from the other connective tissue (skin, achilles tendon), the most part of water is free water and its quantity is increased in the osteoarthrosis. It is supposed that the rearrangement of binding and free-water fractions in the osteoarthrosis is the result of deficiency of hyaluronic acid and therefore this may be regarded in the improvement of methods of treatment. This scientific and methodical approach allow to receive information on the forms and binding energy of water in the biological tissues, which is absorbed from fluids and steam phase and determine characters of the pathological changes.
Clinical observations suggest that estrogens are involved in the pathogenesis of postmenopausal osteoarthritis, but only little is known about the influence of these hormones on articular cartilage cells. The effect of estradiol is mediated by estrogen receptors alpha and beta. The goal of the present study was to search for estrogen receptor alpha in articular tissue from cows, pigs and humans by immunohistochemistry to form a basis for in vitro studies. In addition, we also tried to detect estrogen receptor alpha in cultivated articular chondrocytes from cows and bulls under certain culture conditions. Estrogen receptor alpha is detected by the use of antibody 13H2 in articular chondrocytes from cows, bulls, pigs and humans. Chondrocytes are physiologically exposed to reduced oxygen tension. In isolated articular chondrocytes from cows and bulls incubated either with 21% O2 or with 5% O2 positive cells were also found. These positive results therefore encourage testing the influence of estradiol on cultivated articular cartilage cells in these species under different culture conditions.
To examine the in situ distributions of vitamin D receptors (VDR) and matrix metalloproteinases (MMPs) in osteoarthritic cartilage for comparison with non-arthritic, normal cartilage; and to assess the in vitro effects of 1alpha,25 dihydroxyvitaminD(3)(1alpha,25(OH)(2)D(3)) on MMPs-1, -3 and -9 and prostaglandin E(2)(PGE(2)) production by cultures of human articular chondrocytes (HAC) shown to be VDR-positive.
Using immunohistochemistry VDR expression in different specimens of osteoarthritic cartilage (N=11) was compared to that in normal cartilage (N=6), along with the immunodetection of MMPs-1, -3 and -9. The effects of 1alpha25(OH)(2)D(3)on MMP and PGE(2)production by HAC in vitro, with and without stimulation by TNFalpha or phorbol myristate acetate (PMA), was evaluated using ELISA methodology.
VDR was demonstrated in HAC of all specimens of osteoarthritic cartilage, especially the superficial zone, whereas only two of five normal cartilage specimens were VDR(+)for a minor proportion of HAC. Immunolocalization of MMPs-1, -3 and -9 was often seen in areas where chondrocytes were VDR(+), and dual immunolocalization has demonstrated individual chondrocytes positive for both VDR and MMP-3 in situ. In vitro, 1alpha25(OH)(2)D(3)alone had no effect on MMP-1, -9 and PGE(2)production by HAC, but MMP-3 production was up-regulated by 1alpha25(OH)(2)D(3)either with or without stimulation with TNFalpha or PMA. By contrast the increased production of MMP-9 and PGE(2)induced by PMA was significantly suppressed by concomitant treatment with 1alpha25(OH)(2)D(3).
The demonstration of VDR expression by HAC in osteoarthritic cartilage was often associated with sites where MMP expression was prevalent, observations in contrast to their virtual absence in normal age-matched cartilage. Together with HAC in vitro studies, the data suggests that 1alpha25(OH)(2)D(3)contributes to the regulation of MMP and PGE(2)production by HAC in osteoarthritic cartilage.
Experimental evidence suggests that recommended dosages of some corticosteroids used clinically as antiinflammatory agents for treating arthropathies damage articular cartilage, but low dosages may be chondroprotective. The purpose of this study was to evaluate how different concentrations of methylprednisolone affect chondrocyte function and viability. Articular cartilage and chondrocytes were obtained from young adult horses, 1.5-3.5 years of age. Corticosteroid-induced changes in collagen expression were studied at the transcriptional level by Northern blot analyses and at the translational level by measuring [3H]-proline incorporation into [3H]-hydroxyproline. Fibronectin mRNA splicing patterns were evaluated with ribonuclease protection assays. Cytotoxicity was studied using erythrosin B dye exclusion. Steady-state levels of type II procollagen mRNA decreased without concurrent changes in type I procollagen expression as the medium methylprednisolone concentrations were increased from 1 x 10(1) to 1 x 10(8) pg/ml, dropping below 10% of control values by 1 x 10(5) pg/ml. Cytotoxicity occurred as methylprednisolone levels were increased further from 1 x 10(8) to 1 x 10(9) pg/ml. Changes in total collagen (protein) synthesis were less pronounced, but also demonstrated significant suppression between 1 x 10(4) and 1 x 10(8) pg/ml. Corticosteroid-induced changes in fibronectin isoform levels were evaluated in articular cartilage samples without in vitro culture. The cartilage-specific (V + C)(-) isoform was suppressed in both normal and inflamed joints by a single intraarticular injection (0.1 mg/kg) of methylprednisolone. Combined, these data indicate that methylprednisolone suppresses matrix protein markers of chondrocytic differentiation. Decreased and altered chondrocyte expression of matrix proteins likely contributes to the pathogenesis of corticosteroid-induced cartilage degeneration.
Although osteoarthritis is characterized by a progressive loss of the extracellular cartilage matrix, very little is known about the fate of articular chondrocytes during the progression of the disease. In this study we examined the expression of syndecan-3, a marker of early chondrocyte differentiation, and annexin VI, a marker of late chondrocyte differentiation, in mammalian embryonic growth plate cartilage and normal and osteoarthritic human articular cartilage. Whereas syndecan-3 was expressed in the proliferative and hypertrophic zones of growth platecartilage, immunostaining for annexin VI waspredominately found in the hypertrophic and mineralizing zones of fetal bovine growth plate cartilage. Approximately 20% of chondrocytes were immunopositive for syndecan-3 in normal human articular cartilage, the number of syndecan-3-expressing chondrocytes significantly increased during the progression of osteoarthritis with more than 80% syndecan-3-positive cells in the upper zone of severely affected osteoarthritic cartilage. Similarly, the number of annexin VI-expressing cells significantly increased in the upper cartilage zones during the progression of osteoarthritis. Furthermore, immunostaining for proliferating cell nuclear antigen, a marker for cell proliferation, was detected in chondrocytes in the upper zone of osteoarthritic cartilage. Double-labeling experiments with antibodies against syndecan-3 and annexin VI revealed chondrocytes that expressed only syndecan-3, and cells that expressed both syndecan-3 and annexin VI. These results suggest that the expression of early (proliferating cell nuclear antigen, syndecan-3) and late differentiation markers (annexin VI, alkaline phosphatase) is activated in chondrocytes of osteoarthritic cartilage.
The stability of collagen molecules and moisture capacity of human normal and osteoarthrotic (OA) cartilage were studied before and after extraction of glycosaminoglycans (GAG) by 4M guanidinum chloride. The content and nature of water were determined by Fisher titration, DSC and analysis of sorbtion-desorbtion processes of water vapour in cartilage. The stability of collagen molecules was determined by the degree of enzymatic hydrolysis: collagenase, pronase and pepsin. It was found that weakening of bonds between main compounds of the cartilage matrix and decrease of GAG quantities in the OA cartilage were accompanied by structural disorganization of the collagen network, which is manifested by breakdowns of intramolecular bonds in telopeptides and intermolecular bonds in the spiral part of collagen molecules, these changes may contribute to increase of total water in OA cartilage. The correlation of free and bound water fractions in cartilage was increased from 5 to 44 in OA cartilage. These results can be used as a criterion of pathological condition of human articular cartilage.
The purpose of this work was to test the effect of inhibition of bone remodeling, through the use of the bisphosphonate, zoledronic acid, on cartilage matrix damage in an animal model of cartilage matrix damage.
New Zealand white rabbits were divided into four groups for treatment purposes: (1) untreated controls; (2) injected into one knee joint with the cartilage matrix degradation enzyme, chymopapain; (3) injected into one knee joint with chymopapain and also given subcutaneous injections of the bisphosphonate, zoledronic acid, three times per week until sacrifice at either day 28 or 56 post-chymopapain-injection; (4) received only the zoledronic acid injections. At sacrifice, the knee joints were examined grossly and histologically, and biochemically for proteoglycan content. Urine samples were analysed, at intervals, for levels of collagen cross-links which are biochemical markers of cartilage and bone.
Animals receiving both intraarticular chymopapain injections and subcutaneous zoledronic acid injections displayed a significantly lower degree of grossly and histologically detectable cartilage degeneration on the tibial articular surfaces (the articular surface displaying the greatest degree of degeneration) than did animals only receiving the chymopapain injections. In addition, urinary levels of collagen cross-links for bone and cartilage were significantly higher in those animals only receiving chymopapain injections.
The bone resorption observed after chymopapain injection into the rabbit knee joint can be inhibited through the use of the bisphosphonate, zoledronic acid. Furthermore, zoledronic acid does not increase the level of cartilage degeneration and appears to provide some level of chondroprotection in this model.