The authors have no conflict of interest.
Effects of Estrogen Replacement Therapy on Bone Turnover in Subchondral Bone and Epiphyseal Metaphyseal Cancellous Bone of Ovariectomized Cynomolgus Monkeys†
Article first published online: 15 MAR 2004
Copyright © 2004 ASBMR
Journal of Bone and Mineral Research
Volume 19, Issue 5, pages 823–829, May 2004
How to Cite
Ham, K. D. and Carlson, C. S. (2004), Effects of Estrogen Replacement Therapy on Bone Turnover in Subchondral Bone and Epiphyseal Metaphyseal Cancellous Bone of Ovariectomized Cynomolgus Monkeys. J Bone Miner Res, 19: 823–829. doi: 10.1359/jbmr.040309
- Issue published online: 2 DEC 2009
- Article first published online: 15 MAR 2004
- Manuscript Accepted: 14 JAN 2004
- Manuscript Revised: 13 JAN 2004
- Manuscript Received: 12 AUG 2003
- bone histomorphometry
ERT decreases the severity of OA in OVX cynomolgus monkeys. We show that bone formation is greater in subchondral bone compared with epiphyseal/metaphyseal cancellous bone of the proximal tibia in these animals and that ERT decreases bone formation in both sites. ERT may decrease the risk of OA by decreasing bone formation in the SC bone.
Introduction: Estrogen replacement therapy (ERT) decreases the risk of osteoporosis and osteoarthritis (OA) in postmenopausal women and has been shown to have direct effects on cells of the bone and cartilage. The effects of ERT have been studied extensively in cancellous bone, but subchondral (SC) bone directly beneath the articular cartilage has not been specifically evaluated.
Materials and Methods: Adult feral female cynomolgus monkeys were bilaterally ovariectomized (OVX) to simulate menopause; treated with ERT, soy phytoestrogens (SPE), or no hormones (OVX control group) for 3 years; and labeled with calcein before necropsy. At necropsy, the proximal tibias of 20 randomly selected animals from each treatment group were embedded in bioplastic and sectioned. Areas and labels were measured in a carefully defined region of the SC bone and epiphyseal/metaphyseal cancellous (EMC) bone, and derived dynamic and static indices were compared between the SC and EMC bone and among the three treatment groups. Student's t-tests and ANOVA were used to compare the data.
Results and Conclusions: In both the SC and EMC bone, most of the values for the dynamic indices were highest in the OVX control group, intermediate in the SPE group, and lowest in the ERT group. The mineralizing surface, double-labeled surface, and bone formation rate (surface referent) were significantly higher in the SC bone compared with the EMC bone in the OVX control group. The trabecular bone volume was higher in the SPE-treated group compared with the OVX control group. In conclusion, the bone turnover indices were higher in the SC bone compared with the EMC bone, and ERT decreased these indices in both sites. In addition, SPE was protective against loss of bone volume.
The bone loss that occurs with postmenopausal estrogen deficiency has been extensively investigated, and it is known that estrogen replacement therapy (ERT) decreases the risk of postmenopausal osteoporosis.(1) Studies have shown that estrogen acts directly on osteoclasts to cause increased apoptosis and decreased resorption activity.(2,3) In addition, the presence of estrogen causes a decrease in the number of osteoclast precursor cells(4,5) and decreased levels of osteoclastogenic cytokines.(6–8) Moreover, ERT maintains bone mass and decreases bone turnover in humans and animals.(9,10)
In addition to an increased risk of osteoporosis, epidemiologic evidence suggests that the risk of developing osteoarthritis (OA) also may increase in women after menopause, and most studies suggest that ERT reduces that risk.(11) In a previous study,(12) we demonstrated that articular cartilage lesions of OA in the proximal tibia were reduced by treatment with ERT in ovariectomized (OVX) cynomolgus monkeys with naturally occurring OA. In the current study, bone turnover indices in the subchondral (SC) and epiphyseal/metaphyseal cancellous (EMC) bone of the proximal tibia of these same animals were examined. The animals in this study were relatively young (mean, 11.8 years), had no observed clinical signs of lameness, and had relatively mild histological lesions of OA,(12) thus allowing us to evaluate the changes in SC bone and EMC bone in the early stages of the disease process.
The role of SC bone in the onset and progression of OA is unclear. Some investigators believe that OA is initiated in the cartilage and that cartilage damage leads to SC bone sclerosis, whereas others suggest that OA may be primarilya bone disease, and the thickening and increased density in the SC bone may initiate OA or lead to its progression.(13,14) Data from several animal studies suggest that morphological changes in SC bone may occur before morphological changes in the articular cartilage.(15–17) It also has been demonstrated that several mediators of bone turnover were increased in bone taken from OA femoral heads collected at the time of joint replacement surgery compared with normal control femoral heads.(18) In addition, bone taken from the proximal femoral head (1 cm below and central to the insertion of the ligamentum teres) had increased levels of type I collagen propeptide, alkaline phosphase (ALP), and matrix metalloprotease-2 (MMP-2) compared with bone taken from the distal femoral head (near the head/neck junction),(18) suggesting that OA bone has increased remodeling activity versus normal bone and that this increase is more pronounced in the bone that is in closer proximity to the articular cartilage. Clearly, the role of SC bone in OA pathogenesis warrants further evaluation.
This study used cynomolgus monkeys, which develop naturally occurring OA with bone and articular cartilage lesions that are very similar to the human disease.(17,19) These animals have been used to evaluate the effects of OVX and ERT on the development of osteoporosis(10) and OA.(12) To our knowledge, however, this is the first study to evaluate the effects of OVX and ERT on bone turnover in the SC bone versus EMC bone in any species. Because previous histomorphometry studies have suggested that bony changes within 1-2 mm of the joint are important for the ultimate health of the overlying cartilage,(20–22) SC bone and EMC bone of the proximal tibia were carefully defined and separately evaluated. The study design allowed evaluation of bone turnover activity in an OVX (OVX control) group of monkeys, an OVX ERT group, and an OVX SPE group. Soy phytoestrogens (SPEs) were included in the study because they have been proposed as a possible alternative to ERT.(23,24) The objectives were to evaluate differences in the histomorphometric indices of bone turnover among the three treatment groups and between the SC bone and EMC bone.
MATERIALS AND METHODS
The animals were fed a moderately atherogenic diet (40% of calories from fat) for 26 months to induce lesions of atherosclerosis and were given water ad libitum. At the end of this 26-month period, the monkeys were bilaterally OVX to simulate menopause and were randomly divided into three age- and weight-matched treatment groups: (1) OVX control group (no hormone treatment; n = 60); (2) ERT group (Premarin [conjugated equine estrogens]; Wyeth-Ayerst Laboratories; n = 60); and (3) SPE-treated group (n = 60). An intact, untreated control group was not included in the study design. The treatments were administered in the diet, with Premarin given at an equivalent dose of 0.625 mg/day for women and SPEs given at a dose approximately equivalent to 129 mg/day for women. The treatments were given for 36 months, during which all three groups were fed a moderately atherogenic diet containing 120 kcal/kg of body weight/day (approximately equivalent to 1800 kcal/day for the average woman in the United States).(25)
Plasma estrogen concentrations were measured three times during the 36-month treatment period, and plasma phytoestrogen levels were measured once (34 months after treatment was initiated).(25) All animals received calcein (10 mg/kg) intravenously 14 and 7 days before necropsy.
Necropsy and tissue preparation
At necropsy, both knee joints were collected. A midcoronal section of the right proximal tibia, including both medial and lateral tibial plateaus from 20 randomly selected animals in each treatment group, was embedded in Bioplastic (Wards Scientific, Rochester, NY, USA). Ten-micrometer sections were cut using a sledge microtome and mounted unstained on glass slides using Eukitt (Calibrated Instruments, Hawthorne, NY, USA) mounting material. The slides were randomized to blind the evaluator to treatment group.
Measurements were made using the Osteomeasure histomorphometry system (Osteometrics, Atlanta, GA, USA) by tracing the perimeter of the tissue using a digitized pad. Measurements of the SC bone were taken from the medial tibial plateau in a 3.5-mm-wide field, beginning 2 mm medial to the central long axis (Fig. 1A), using a light microscope with a 2× objective. SC bone was defined as the bone between the calcified cartilage-bone junction and the marrow space. At the lower limit of the SC bone, trabeculae that were narrower (vertically) than they were wide (horizontally) were not included. Areas of bone that were below a void space were included if the SC bone below the void was of greater thickness than the void space (Fig. 1A). The void spaces were also traced, and the area of the void was subtracted from the total area to give the bone area. Calcein labels were measured under fluorescent microscopy using a 10× objective in the same 3.5-mm field.
Trabecular bone measurements were taken in a 3.5 × 3.5 mm field 3 mm below the lower limit of the SC bone, centered on the central long axis (Fig. 1B). This was the largest field that would accommodate the smallest tibia in the study. Measurements of the trabecular bone were taken using a 2× objective under light microscopy by tracing the perimeter of the bone. Void spaces that were enclosed by bone were included and the void spaces were later traced and subtracted from the total bone area. Measurements of trabecular bone calcein labels were taken using a 10× objective under fluorescent light in the same 3.5 × 3.5 mm field.
The primary measurements, formulas for derived indices, and abbreviations are listed in Table 1(10) and are based on the recommendations of the American Society for Bone and Mineral Research nomenclature committee.(26)
The means and SE are presented in the original scale; however, the analyses for most of the variables were performed using square root transformation. The variables that were normally distributed and did not require square root transformation were bone surface to bone volume ratio (BS/BV), bone volume (BV/TV), and trabecular number (Tb.N). The three treatment groups were compared using ANOVA and posthoc tests (Tukey method). The results for the SC bone were compared with the EMC bone using paired t-tests. p values less than 0.05 were considered statistically significant.
Plasma hormone concentrations revealed increased 17β-estradiol levels in the Premarin group (mean for OVX control = 31.6 pM; Premarin group = 517.2 pM; SPE group = 25.7 pM) that were stable across time, as well as detectable SPE levels in the SPE group (mean, 776 nM).(25) There was significant weight gain among the animals during the 3-year study (mean weight gain, 0.5 kg), but there were no significant differences in weight among the three treatment groups at the beginning or termination of this study (Table 2). Also, there was no significant increase in trunk length over the 3-year treatment period, and no difference in trunk length or age among the three treatment groups (Table 2).
In both the SC and EMC bone, nearly all of the values (with the exception of the mineral apposition rate [MAR] in the EMC bone and the BS/BV in both sites) for the dynamic indices were the highest in the OVX control group, intermediate in the SPE group, and lowest in the ERT group. In the SC bone, labels were present primarily on the bone margins; however, there were labels present along the calcified cartilage junction in two of the samples.
The MAR was significantly lower in the SC bone of the ERT group (p = 0.01) compared with the OVX control animals (Fig. 2A).
The mineralizing surface (MS/BS) was significantly lower in the SC (p = 0.004) and EMC (p = 0.019) bone of the ERT group compared with the OVX control group (Fig. 2B). In addition, the MS/BS was significantly higher in the SC bone compared with the EMC bone in the OVX control (p = 0.008) and SPE (p = 0.009) groups.
The double-labeled surface (dLS/BS) was significantly lower in the SC (p = 0.005) and EMC (p = 0.002) bone of the ERT group compared with the OVX control group (Fig. 2C). Also, the dLS/BS was significantly higher (p = 0.008) in the SC bone compared with the EMC bone in the OVX control group.
The bone formation rate (bone surface referent; BFR/BS) was significantly lower in the SC (p = 0.005) and EMC (p = 0.011) bone of the ERT group compared with the OVX control group (Fig. 2D). In addition, the BFR/BS was significantly higher (p = 0.014) in the SC bone compared with the EMC bone in the OVX control group.
The bone formation rate (bone volume referent; BFR/BV) was significantly lower in the SC (p = 0.006) and EMC (p = 0.025) bone of the ERT animals compared with the OVX control animals (Fig. 2E).
Static histomorphometry—subchondral bone
There were no significant differences among the three treatment groups for SC bone width or area (Table 3).
Static histomorphometry—epiphyseal/metaphyseal cancellous bone
The Tb.N was significantly higher in the ERT (p = 0.007) and SPE (p = 0.003) animals compared with the OVX control animals (Tb.N; Fig. 3A). In addition, the trabecular separation was significantly lower in the ERT (p = 0.007) and SPE (p = 0.003) group compared with OVX control animals (Tb.Sp; Fig. 3B). The trabecular bone thickness was significantly lower in the ERT group (p = 0.036) compared with the OVX control group (Tb.Th; Fig. 3C). Bone volume was significantly higher in the SPE group (p = 0.028) compared with the OVX control animals (BV/TV; Fig. 3D).
In this study of early OA in nonhuman primates, the indices of bone turnover were greater in the SC bone than the EMC bone in all three treatment groups, and these differences were most pronounced in the estrogen-deficient OVX control group. This is the first study to show an increase in bone turnover indices in SC bone compared with EMC bone and may provide insight into how estrogen may be protective against the development of OA. We hypothesize that estrogen deficiency may lead to an increased risk of developing OA by causing alterations in SC bone remodeling, and subsequently, bone structure. This may lead to changes in load distribution across the tibial plateaus, potentially causing cartilage damage and osteophyte formation.
SC bone is morphologically more similar to cortical bone than trabecular bone. In most studies, cortical bone is less sensitive to estrogen withdrawal than trabecular bone(27,28); however, some studies have shown the opposite results with cortical bone being more sensitive to estrogen withdrawal(29) or more sensitive to the lack of estrogen receptor-β.(30) In this study, the indices of bone turnover were higher in SC bone compared with EMC bone and were significantly higher in the absence of estrogen. It seems that, despite the morphological similarities to cortical bone and its close proximity to trabecular bone, SC bone must be regarded as a separate entity, with characteristics and activities that are unique.
There are differences between SC bone and EMC bone that may explain the differences seen in the indices of bone turnover. Previous studies have shown that load increases bone turnover,(31–33) and EMC bone, located deeper in the epiphysis, may bear less of the biomechanical load compared with SC bone of the proximal tibia. Another explanation for a higher turnover in SC bone may be the interaction with the overlying cartilage. It has been demonstrated that articular cartilage chondrocytes produce growth factors such as insulin-like growth factor-1,(34) transforming growth factor-β,(35) and vascular endothelial growth factor (VEGF),(36) and it has been shown that estrogen and pregnancy may modulate the production and activity of growth factors in cartilage.(37,38) All of these growth factors, and perhaps others, have the potential to influence the activity of the underlying bone, and recent studies involving VEGF show that this is a likely possibility.(36,39,40)
In addition to the differences seen in the dynamic histomorphometric indices between the two locations, there were also differences among the treatment groups within SC bone and EMC bone. ERT, but not SPEs, significantly decreased all indices of bone turnover in both sites (except MAR in EMC bone, which was decreased but did not reach statistical significance). This result was not surprising, because it has been established that ERT decreases bone turnover in both monkeys and humans(1,10) and decreases the risk of developing osteoporosis in postmenopausal women.(1)
In this study, there were no significant differences in SC bone thickness or area among the three treatment groups. In a previous study, in a larger group of animals that included the animals from this study, there also were no significant differences in SC bone thickness or area among the three treatment groups.(12) However, when all of the data (articular cartilage, calcified cartilage, and SC bone grades and measurements) were summarized using factor analysis, the factor that was loaded by SC bone thickness and area was significantly higher in the ERT group compared with the OVX control group. This was most likely because of the effects of estrogen in decreasing bone turnover and preserving bone mass.
As expected, there was a significantly greater trabecular number and lower trabecular separation in the EMC bone with ERT. In addition, the bone volume in the ERT group was increased in comparison with the OVX control group, but surprisingly, this difference was not statistically significant. Because estrogen is known to maintain bone volume, we would expect to see a significant decrease in bone volume in the estrogen-deficient OVX control group. The explanation for this result is unclear. In a previous histomorphometry study of bone turnover indices in cynomolgus monkeys, however, it was determined by power calculation that the minimal sample size required per treatment group to see statistically significant differences between groups for static histomorphometry was 29 (β = 0.6) and for dynamic histomorphometry was 19.(10) This study included 20 samples per treatment group; thus, the static histomorphometry arm of the study was somewhat underpowered.
The results for the derived static indices in the SPE group EMC bone were also surprising. The bone volume in the SPE group was significantly higher than the OVX control group, and SPE treatment significantly increased the trabecular number and decreased the trabecular separation. These results combined with the results of the dynamic histomorphometry data (showing that SPE had an intermediate effect between ERT and OVX control, although not statistically different from either group) indicate that SPE treatment, although not as effective as ERT, may be beneficial in preventing bone loss in postmenopausal women. This also supports the results of several previous studies showing that SPEs conserve bone mass in OVX rats and may have similar effects in higher mammalian species.(41)
The results of this study provide new insight into the properties of SC bone versus EMC bone of the proximal tibia and the response of these sites to ERT and SPE treatment in early OA. The results of this study clearly show that there is a higher bone turnover in SC bone compared with EMC bone and that bone turnover in both sites is decreased by ERT in estrogen-deficient animals. Furthermore, it also seems that SPEs have an effect on preserving bone volume in the absence of estrogen.
The authors thank Jessika Stadden for technical assistance. We also thank Dr Thomas Clarkson for providing the knee joints for this study, Dr Mary Anthony for providing the details of the clinical trial, and Jean Gardin for assistance with tissue collection. This study was supported by National Institutes of Health Grant RR14099 and the University of Minnesota Doctoral Dissertation Fellowship.
- 12001 Sex steroids and bone. Physiol Rev 81:419–447.
- 21997 Estrogen inhibits bone resorption by directly inducing apoptosis of the bone-resorbing osteoclasts. J Exp Med 186:489–495., , , , , , , , , , , ,
- 31996 Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-β. Nat Med 2:1132–1134., , , , ,
- 41998 Ovariectomy augments B lymphopoiesis and generation of monocyte-macrophage precursors in rat bone marrow. Am J Physiol 274:E476–E483., , ,
- 51996 Estrogen modulates the recruitment of myelopoietic cell progenitors in rat through a stromal cell-independent mechanism involving apoptosis. Blood 87:2683–2692.,
- 61995 Mechanisms of disease: Bone marrow, cytokines, and bone remodeling—emerging insights into the pathophysiology of osteoporosis. N Engl J Med 332:305–311.,
- 71996 Estrogen, cytokines, and pathogenesis of postmenopausal osteoporosis. J Bone Miner Res 11:1043–1051.
- 81994 Skeletal effects of estrogen. Endocr Rev 15:275–300., ,
- 92000 Prevention of postmenopausal osteoporosis with oestrogen replacement therapy and associated compounds: Update on clinical trials since 1995. Hum Reprod 6:419–426.,
- 101994 Bone functional changes in intact, ovariectomized, and ovariectomized, hormone-supplemented adult cynomolgus monkeys (Macaca fascicularis) evaluated by serum markers and dynamic histomorphometry. J Bone Miner Res 9:527–540., , , , , ,
- 111999 Estrogen and osteoarthritis: How do we explain conflicting study results? Prev Med 28:445–448.,
- 122002 Effects of long-term estrogen replacement therapy on osteoarthritis severity in cynomolgus macaques. Arthritis Rheum 46:1956–1964., , ,
- 132001 The role of bone in osteoarthritis. Age Ageing 30:374–378., ,
- 141986 Role of subchondral bone in the initiation and progression of cartilage damage. Clin Orthop 213:34–40.,
- 151994 Spontaneous joint degeneration in the guinea pig knee studied by magnetic resonance imaging. Br J Rheumatol 33(Suppl 1):354., , ,
- 161994 Evidence for cathepsin B involvement in subchondral bone changes during early natural osteoarthritis in the guinea pig. Br J Rheumatol 33(Suppl 1):169., , ,
- 171994 Osteoarthritis in cynomolgus macaques: A primate model of naturally occurring disease. J Orthop Res 12:331–339., , , , ,
- 181998 Abnormal cancellous bone collagen metabolism in osteoarthritis. J Clin Invest 101:1596–1603.,
- 191996 Osteoarthritis in cynomolgus macaques. III: Effects of age, gender, and subchondral bone thickness on the severity of disease. J Bone Miner Res 11:1209–1217., , , ,
- 201991 Osteoarthritic changes in canine articular cartilage, subchondral bone, and synovium fifty-four months after transection of the anterior cruciate ligament. Arthritis Rheum 34:1560–1570., , ,
- 211984 Finite element studies of some juxtarticular stress changes due to localized subchondral stiffening. J Biomech 17:11–24., , ,
- 221989 Gait alterations in dogs after transection of the anterior cruciate ligament. Arthritis Rheum 32:1142–1147., , , ,
- 232001 A review of the evidence for the use of phytoestrogens as a replacement for traditional estrogen replacement therapy. Arch Intern Med 161:1161–1172.,
- 241994 Interaction of naturally occurring nonsteroidal estrogens with expressed recombinant human estrogen receptor. J Steroid Biochem Mol Biol 49:153–160.
- 252001 Inhibition of postmenopausal atherosclerosis progression: A comparison of the effects of conjugated equine estrogens and soy phytoestrogens. J Clin Endocrinol Metab 86:41–47., ,
- 261987 Bone histomorphometry: Standardization of nomenclature, symbols and units. J Bone Miner Res 2:595–610., , , , , , ,
- 271999 Effects of menopause on age-dependent bone loss on the axial and appendicular skeletons in healthy Japanese women. Osteoporos Int 10:377–383., , , ,
- 282002 Mesenchymal stem cells and osteoblast differentiation. In: BilezikianJP, RaiszLG, RodanGA (eds.) Principles of Bone Biology. Academic Press, New York, NY, USA, p. 68.,
- 291997 The androgenic anabolic steroid nandrolone decanoate prevents osteopenia and inhibits bone turnover in ovariectomized cynomolgus monkeys. Bone 20:355–364., , , , , ,
- 301999 Increased cortical bone mineral content but unchanged trabecular bone mineral density in female ER β-/- mice. J Clin Invest 104:895–901., , , ,
- 312002 Skeletal adaptations to alterations in weight-bearing activity: A comparison of models of disuse osteoporosis. Sports Med 32:459–476.,
- 321999 Mechanical signaling in the development of postmenopausal osteoporosis. Lupus 8:388–392.
- 331999 Trabecular bone turnover and bone marrow cell development in tail-suspended mice. J Bone Miner Res 14:1596–1604., , , , , , ,
- 341998 IGF/IGFBP axis in cartilage and bone in osteoarthritis pathogenesis. Inflamm Res 47:90–100., , ,
- 352002 Recent advances in TGB-β effects on chondrocyte metabolism potential therapeutic roles of TGB-β in cartilage disorders. Cytokine Growth Factor Rev 13:241–257., ,
- 362002 Development and regulation of osteophyte formation during experimental osteoarthritis. Osteoarthritis Cartilage 10:180–187., , , , ,
- 372000 Functional estrogen receptors in adult articular cartilage: Estrogen replacement therapy increases chondrocyte synthesis of proteoglycans and insulin-like growth factor binding protein 2. Arthritis Rheum 43:2081–2090., , , ,
- 381998 Influence of pregnancy on gene expression in rabbit articular cartilage. Osteoarthritis Cartilage 6:341–350., ,
- 392001 The splice variants VEGF121 and VEGF189 of the angiogenic peptide vascular endothelial growth factor are expressed in osteoarthritic cartilage. Arthritis Rheum 44:1082–1088., , ,
- 402002 Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci USA 99:9656–9661., , , , , , , , , , , , ,
- 411998 Phytoestrogens and bone. Clin Endocrinol (Oxf) 12:543–557.,