Ru-Chun Dai

A Comparison Study of the Reference Curves of Bone Mineral Density at Different Skeletal Sites in Native Chinese, Japanese, and American Caucasian Women

To understand the differences among reference curves for bone mineral density (BMD) for Chinese, Japanese, and American Caucasian women, we measured the BMD at the anteroposterior (AP) lumbar spine (L1–L4), lateral lumbar spine (L2–L4), hip (including the femoral neck, trochanter, intertrochanter, Ward’s triangle, and total hip), and ultradistal forearm by the dual-energy X-ray absorptiometry (DXA) in a total of 2728 healthy Chinese women, aged 5–96 years. Documented BMD data for Japanese women and device manufacturer’s BMD new reference databases (including the NHANES III dataset) for American Caucasian women were also used in this study. The cubic regression model was found to fit best in analyzing the age-associated variations of BMD at various sites in Chinese women, i.e., the equations had the largest coefficient of determination (R2). At the AP/Lat spine, trochanter, intertrochanter, and Ward’s triangle, BMD reference curves for Chinese women were lower than those for Japanese or Caucasian women, while at the femoral neck, total hip, and ultradistal forearm, the reference curves for Chinese women were higher than those for Japanese women, with overlaps and crossing of the curves for some age spans in comparing the Chinese and Caucasian women. There were significant differences in the peak BMD (PBMD) at various sites among the Chinese, Japanese, and Caucasian women (P = 0.000). The PBMDs for Chinese women at the lumbar spine and various sites of the hip were 5.7% ± 2.1% (mean ± SD, range, 2.7–7.9%) lower than those for Japanese women and 5.1% ± 2.7% (range, 0.5–7.2%) lower than those for Caucasian women; however, the PBMDs for Chinese women were 26.2% higher than those for Japanese women and 10% higher than those for Caucasian women at the ultradistal forearm. After the PBMD, average T-scores of Chinese women for losses at the AP lumbar spine with increasing age were nearly identical to those for Japanese women, but both were greater than those for Caucasian women. The average T-scores for BMD loss at various sites in Chinese women were higher than those for both Japanese and Caucasian women except at the femoral neck, where the T-scores of Chinese women were exceeded by those of both Japanese and Caucasian women. Estimated from the T-score curve of BMD loss, the age of osteoporosis occurrence at the femoral neck in Chinese women was about 10 years later than that in Japanese or Caucasian women; at the AP spine, Chinese women were similar to Japanese women; at the other sites, the age for occurrence of osteoporosis in Chinese women was about 5–15 years earlier than that in either Japanese or Caucasian women. There are differences in prevalence or odds ratio (OR) of osteoporosis at the same skeletal region for Chinese, Japanese, and Caucasian women aged ≥50 years or at different skeletal regions in women of the same race. The prevalences of osteoporosis at various regions of the hip in Chinese women are 10.1–19.8% and ORs are 22.0–32.3, of which prevalence at the femoral neck is the lowest (10.1%); the prevalences of osteoporosis in Japanese women are 11.6–16.8% and ORs are 21.1–26.3, of which prevalence at the femoral neck is the lowest (11.6%); and the prevalences of osteoporosis in Caucasian women are 13.0–20.0% and ORs are 19.4–48.9, of which prevalence at the femoral neck is the highest (20%). In conclusion, racial differences in BMD reference curves, prevalences, and risks of osteoporosis at various skeletal sites exist among native Chinese, Japanese, and American Caucasian women.

Application of micro-ct assessment of 3-d bone microstructure in preclinical and clinical studies

As the mechanical competence of trabecular bone is a function of its apparent density and 3-D distribution, assessment of 3-D trabecular structural characteristics may improve our ability to understand the pathophysiology of osteoporosis, to test the efficacy of pharmaceutical intervention, and to estimate bone biomechanical properties. We have studied ovariectomy-induced osteopenia in rats and its treatment with agents such as estrogen and sodium fluoride. We have demonstrated that 3-D micro-computed tomography (µCT) can directly quantify mouse trabecular and cortical bone structure with an isotropic resolution of 6 µm3. µCT is also useful for studying osteoporosis in mice and phenotypes of mice with gene manipulation, such as SHIP-knockout mice, which are severely osteoporotic due to increased numbers of hyperresorptive osteoclasts, PTHrP heterozygous-null mice, and mice with Zmpste24 deficiency. µCT can quantify osteogenesis in mouse Ilizarov leg-lengthening procedures, osteoconduction in a rat cranial defect model, and structural changes in arthritic rabbits, rats, and mice. In clinical studies, we evaluated longitudinal changes in the iliac crests. Paired bone biopsies from the same premenopausal and postmenopausal women showed the changes in 3-D trabecular structure, such as decreased trabecular thickness, shifting of trabecular model from platelike structure to rodlike structure, and decreased degree of anisotropy were remarkable. Treatment with PTH in postmenopausal women with osteoporosis significantly improved trabecular morphology with a shift toward a more platelike structure, increased trabecular connectivity density, and increased cortical thickness. Paired bone biopsy specimens from the iliac crest in postmenopausal women with osteoporosis before and an average of 2 years after beginning of estrogen replacement therapy demonstrated that posttreatment biopsies showed a significant change in the ratio of plates to rods and statistically insignificant changes in other 3-D trabecular parameters. Thus, µCT can characterize 3-D structure of various animal models, and the longitudinal changes in 3-D bone microarchitectural integrity that deteriorates in the transmenopausal period, is preserved with HRT, and is improved with PTH treatment in postmenopausal women.

miR-148a regulates osteoclastogenesis by targeting V-maf musculoaponeurotic fibrosarcoma oncogene homolog B.

MicroRNAs (miRNAs) play crucial roles in bone metabolism. In the present study, we found that miR‐148a is dramatically upregulated during osteoclastic differentiation of circulating CD14+ peripheral blood mononuclear cells (PBMCs) induced by macrophage colony stimulating factor (M‐CSF) and receptor activator of nuclear factor‐κB ligand (RANKL). Overexpression of miR‐148a in CD14+ PBMCs promoted osteoclastogenesis, whereas inhibition of miR‐148a attenuated osteoclastogenesis. V‐maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) is a transcription factor negatively regulating RANKL‐induced osteoclastogenesis. miR‐148a directly targeted MAFB mRNA by binding to the 3′ untranslated region (3′UTR) and repressed MAFB protein expression. In vivo, our study showed that silencing of miR‐148a using a specific antagomir‐inhibited bone resorption and increased bone mass in mice receiving ovariectomy (OVX) and in sham‐operated control mice. Furthermore, our results showed that miR‐148a levels significantly increased in CD14+ PBMCs from lupus patients and resulted in enhanced osteoclastogenesis, which contributed to the lower bone mineral density (BMD) in lupus patients compared with normal controls. Thus, our study provides a new insight into the roles of miRNAs in osteoclastogenesis, and contributes to a new therapeutic pathway for osteoporosis.

Establishment and evaluation of bone mineral density reference databases appropriate for diagnosis and evaluation of osteoporosis in Chinese women

Abstract. This study was designed to establish Bone Mineral Density (BMD) Reference Databases for multiple skeletal sites appropriate for the diagnosis and evaluation of osteoporosis (OP) in Chinese women. We recruited 2702 healthy Chinese women, 5–96 years of age, for BMD assessment. BMD values at multiple skeletal sites including anteroposterior (AP) and lateral (Lat) lumbar spine, hip, and forearm were measured by dual-energy X-ray absorptiometry (DXA) using a QDR 4500A device; results were analyzed according to age group using eight regression models. BMD Reference Databases (CWD) were established according to the best regression equation and compared with Hologic reference databases for “Oriental Women” (OWD). Results indicated that the cubic regression model was superior to the quadratic, linear, logarithmic, and exponential regression models, etc. for our purpose, with a determinate coefficient (R2) of 0.363–0.650 (P = 0.000). We included 1636 female patients, aged 35–86 years, in our tests. In comparison with Hologic Reference Databases, the mean detection rate of OP in the newly established BMD Reference Databases for Chinese Women (CWD) was 16.0% ± 2.68% lower (range, 13.7%–20.5%) at the AP spine, 16.8% ± 11.0% lower (range, 3.5%–32.8%) at the Lat spine (except for L4), 18.7% ± 4.6% lower (range, 12.6%–24.2%) at the hip, and 14.3% ± 6.9% higher (range, 4.7%–24.3%) at the forearm. The difference in detection rates for OP was significant between the two reference databases (P = 0.000), which was consistent with the differences in peak BMD values and the biological variability between them. Based upon our data, we confirmed that the Hologic BMD Reference Databases for Oriental Women (OWD) were not suitable for the diagnosis of OP in Chinese women; the BMD Reference Databases for Chinese Women (CWD) established in this study would provide reliable diagnostic standards for detection of OP in the women of South China.

Effects of genistein on vertebral trabecular bone microstructure, bone mineral density, microcracks, osteocyte density, and bone strength in ovariectomized rats

Until now, the effects of phytoestrogen on bone in both women and ovarian hormone-deficient animal models of osteoporosis have remained uncertain. We have aimed here to investigate the effect of genistein (GEN) on trabecular bone quality in ovariectomized (OVX) rats. Forty 7-month-old female Sprague-Dawley rats were randomly divided into the following four groups: OVX, sham-operated (SHAM), treated with 17β-estradiol (EST, 10 μg·kg−1·day−1), and GEN (5 mg·kg−1·day−1). At 15 weeks postoperation, the compressive test was performed on the L5 vertebral body; additionally, microcomputed tomography (μ-CT) assessment was performed to estimate the bone mineral density (BMD) and microstructure parameters of the L6 vertebral body. After fatigue damage testing, the L6 vertebral body was bulk-stained in 1% basic fuchsin and embedded in methylmethacrylate. The L4 vertebral body was embedded in methylmethacrylate for dynamic histomorphometry analysis without staining. Mounted bone slices were used to measure microcrack parameters, empty osteocyte lacuna density (e.Lc.Dn), and osteocyte density (Ot.N/T.Ar). Maximum loading (ML) and Ot.N/T.Ar were significantly lower in the OVX group than in the other groups. E.Lc.Dn was significantly decreased in GEN and EST groups compared to the OVX group. ML was significantly decreased in the GEN group compared to the SHAM group. Microcrack density, microcrack surface density, and microcrack length were significantly increased in the OVX group compared to the other groups. Mineral apposition rate was significantly decreased in the OVX group compared to the SHAM and GEN groups. Bone formation rate was significantly decreased in the OVX group compared to other groups. There were no significant differences with regard to mineralizing surface among the four groups. Volumetric BMD at organ was significantly lower in OVX, EST, and GEN groups than in the SHAM group. Bone mineral content was significantly lower in the OVX group than in the SHAM group. Bone volume fraction and trabecular number were significantly decreased in OVX, EST, and GEN groups compared to the SHAM group. Structure model index was significantly lower in the SHAM group than in OVX, EST, and GEN groups. Trabecular separation was significantly increased in the OVX group compared to SHAM and EST groups. There were no significant differences with regard to the trabecular thickness (Tb,Th) between SHAM, GEN, and OVX groups. Tb.Th was significantly lower in the EST group than in the SHAM group. Connectivity density (Conn.D) was significantly lower in the OVX group than in SHAM and GEN groups, and Conn. D was significantly lower in the EST group than in GEN. In conclusion, the present study demonstrates that GEN preserved the biomechanical quality of the trabecular bone regardless of the microstructure and BMD.

Determination of age-specific bone mineral density and comparison of diagnosis and prevalence of primary osteoporosis in Chinese women based on both Chinese and World Health Organization criteria

The aim of this study was to determine age-specific bone mineral density (BMD) at various skeletal regions in a native Chinese reference population, and to explore the differences in the diagnosis of primary osteoporosis and estimated prevalence of osteoporosis based on both Chinese criteria (BMD of subjects, 25% lower than the peak BMD) and WHO criteria (BMD of subjects, 2.5 SD [T-score ≤ −2.5] lower than the young adult mean [YAM]). There were 3406 subjects in our female reference population, ranging in age from 10 to 90 years. A dual-energy X-ray absorptiometry (DXA) fan-beam bone densitometer (Hologic QDR 4500A) was used to measure the BMD in subjects at the posteroanterior (PA) spine (L1–L4), supine lateral spine (L2–L4 including areal BMD [aBMD] and volumetric BMD [vBMD]), hip (including femoral neck and total hip), and radius + ulna ultradistal (R + UUD) of the forearm. Cross-sectional data analysis in stratified 5-year age intervals revealed that the peak BMD (PBMD) at various skeletal regions occurred within the age range of 30–44 years, with PBMD at the lateral spine and femoral neck occurring at 30–34 years, posteroanterior spine and total hip at 35–39 years, and ultradistal forearm at 35–44 years. The reference values of BMD (PBMD) calculated using Chinese criteria for the diagnosis of primary osteoporosis were significantly higher than the young adult mean (YAM) using WHO criteria for all skeletal regions except for the total hip, at a range of 0.9%–3.8% higher. The BMD cutoff values using Chinese criteria for the diagnosis of osteoporosis were 3.7%–10.9% higher than those using WHO criteria for various skeletal regions. The prevalence rate of primary osteoporosis according to Chinese criteria in subjects ranging from 50 to 90 years was 41.5% at the PA spine, 53.9% at the lateral spine, 34.2% at the femoral neck, 30.7% for total hip, and 51.4% at R + UUD; while according to WHO criteria, this rate was 32.1% at the PA spine, 34.9% at the lateral spine, 16.3% at the femoral neck, 18.9% for total hip, and 45.2% at R + UUD. The prevalence of primary osteoporosis according to both criteria varied with the age and skeletal region of the subjects. The prevalence of primary osteoporosis using Chinese criteria, compared with WHO criteria was 31% higher at the lumbar spine, 109% higher at the femoral neck, and 14% higher at the ultradistal forearm. In conclusion, PBMD occurs in the age range of 30–44 years in native Chinese females. The BMD reference values, BMD cutoff values, and prevalence of primary osteoporosis determined by Chinese criteria are all higher than those determined by the WHO criteria; thus, the application of Chinese criteria may overestimate the number of patients with primary osteoporosis.

Regionally specific compensation for bone loss in the tibial trabeculae of estrogen-deficient rats

Background: Bone mineral density (BMD) and microstructural variations have been extensively investigated in recent years; however, the compensation for bone loss between different regions is still unclear. Purpose: To fully characterize regional variations in bone mineral density (BMD) as well as the microstructure and dynamic changes of rat tibial trabeculae that occur with bone loss associated with estrogen deficiency. Material and Methods: Female Sprague-Dawley rats were ovariectomized (OVX), sham-operated (sham), or left unoperated (baseline control). The left tibiae were harvested at baseline, and at postoperative weeks 3 and 15. High-resolution micro-computed tomography (µCT) was used to identify the densitometric and microstructural properties of trabeculae in the proximal ends of the rat tibia, specifically the epiphysis and metaphysis. Results: Volumetric BMDs at the organ (organ BMD) and tissue (tissue BMD) levels were significantly higher for trabeculae at the epiphysis than metaphysis. Moreover, trabeculae at the epiphysis were thicker, and fewer in number and connectivity than those at the metaphysis, which were more rod like. Trabeculae at the metaphysis were more susceptible to bone loss induced by estrogen deprivation than at the epiphysis, and the regions varied greatly in their adaptation to this loss. At the metaphysis, trabecular tissue BMD and thickness were unexpectedly higher at postoperative week 15 than week 3 or baseline. In contrast, at the epiphysis, tissue BMD did not change with time, but trabecular thickness significantly increased at week 15 compared to baseline and was also greater in OVX compared to sham rats. Conclusion: Metaphyseal and epiphyseal trabeculae show regionally specific variations in BMD and microstructure. The former are more susceptible to bone loss induced by estrogen deficiency and would be strengthened by either hypertrophy or hypermineralization, while epiphyseal trabeculae are mainly strengthened by thickening.

Effects of 17 β-estradiol on the expression of interstitial collagenases-8 and −13 (MMP-8 and MMP-13) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in ovariectomized rat osteoblastic cells

Estrogen plays an important role in maintaining normal bone metabolism via the direct or indirect regulation of bone cells. Osteoblastic cells, as the target cells of estrogen, can secrete multiple matrix metalloproteinases (MMPs) that participate in bone remodeling. It has been demonstrated that bone loss induced by estrogen deficiency is closely related to the abnormal expression of multiple MMPs in osteoblastic cells. However, the regulating action of estrogen on the expression of interstitial collagenases MMP-8 and MMP-13 in osteoblastic cells in vivo remains unclear. We used an ovariectomized osteoporotic rat model to analyze the changes in the histomorphometric parameters of bone after and without treatment with 17β-estradiol (E2); We also used immunohistochemistry and in situ hybridization to observe changes in the expression of mRNA and the proteins MMP-8, MMP-13 and TIMP-1 in osteoblastic cells in rat proximal tibia. In this study, we found that in the ovariectomized rat the expression of MMP-13 mRNA and protein increased markedly, whereas the expression of MMP-8 and TIMP-1 mRNA and protein did not change significantly. Our analysis showed that the expression of MMP-13 protein was correlated positively to bone trabecular separation, osteoid surface area, and negatively to trabecular numbers and the percentage of trabecula bone volume/total tissue volume. Our results suggest that MMP-13 plays an important role in estrogen deficiency-induced bone loss, while estrogen can inhibit bone resorption and reduce bone turnover rate by down-regulating the expression of MMP-13 in osteoblastic cells.

Quantitative associations between osteocyte density and biomechanics, microcrack and microstructure in OVX rats vertebral trabeculae.

Osteocytes actively regulate bone modeling and remodeling, direct skeletal mineralization, and regulate calcium/phosphate homeostasis and extracellular matrix metabolism; yet the specific role of osteocytes in maintaining bone structural integrity and strength is unknown. Studies have shown that the density of osteocytes decreases with age and estrogen deficiency, as seen in postmenopausal women. Here, we examined the relationships between osteocyte density and the related variables, including biomechanics, bone mineral density, microcrack and microstructure of vertebral trabeculae, in ovariectomized rats. We found that osteocyte density correlated with some of the parameters that determine the biomechanical quality of bone. Our findings suggest that osteocytes could play a crucial role in maintaining the mechanical quality of bone, and osteocyte density could be considered as an alternative index in assessing bone quality.

Microcracks: an alternative index for evaluating bone biomechanical quality

To investigate microcracks as a potential index of bone biomechanical quality in rats, 98 Sprague-Dawley (SD) rats, 10 months old, were used. Eight rats were killed at the beginning of the study, to serve as baseline controls. The remaining 90 rats were ovariectomized (OVX), and treated with 17 Β-estradiol (EST) at 10 µg/kg per day, or sham-operated (sham). These 90 rats were killed at 3, 15, and 12 weeks post-surgery. Bone mineral density (BMD) of the total body, the lumbar spine L1–L4, and the tibiae was measured, using dual-energy X-ray absorptiometry (DXA). Bone histomorphometry of the right proximal tibial metaphysis was performed. Compressive testing was performed on the L5 vertebral body. Microcrack density (CrDn) and microcrack surface density (CrSDn) of L4 vertebral bodies that were fatigue-damaged in vitro were determined from bone sections. BMD at various sites, and CrDn and CrSDn were higher at weeks 15 and 21 than at week 3 post-operation. Trabecular separation (TbSp) increased, while trabecular number (TbN) decreased, and the maximum loading (ML) and elastic modulus (EM) of the vertebrae reached their peak values at week 15. At week 3 post-surgery, OVX rats displayed greater TbSp, CrDn, and CrSDn but less trabecular bone volume (BV) than both sham and EST rats. At week 15, BMD and ML were decreased in OVX rats compared with sham rats. At week 21, BMD, TbN, and TbAr were decreased in OVX compared with EST and sham rats. The OVX rats showed greater TbSp, bone formation rate, mineral apposition rate, percent labeled perimeter (%Pm), CrDn, and CrSDn, and lower ML and EM values than both EST and sham rats. Thus, microcrack parameters represent bone biomechanical quality changes associated with ovariectomy in rats, and they indicate the efficacy of estrogen replacement therapy.