The Protective Effect of Cordycepin On Alcohol-Induced Osteonecrosis of the Femoral Head.

BACKGROUND: Alcohol abuse is known to be a leading risk factor for atraumatic osteonecrosis of the femoral head (ONFH), in which the suppression of osteogenesis plays a critical role. Cordycepin benefits bone metabolism; however, there has been no study to determine its effect on osteonecrosis. METHODS: Human bone mesenchymal stem cells (hBMSCs) were identified by multi-lineage differentiation. Alkaline phosphatase (ALP) activity, RT-PCR, western blots, immunofluorescent assay and Alizarin red staining of BMSCs were evaluated. A rat model of alcohol-induced ONFH was established to investigate the protective role of cordycepin against ethanol. Hematoxylin & eosin (H&E) staining and micro-computerized tomography (micro-CT) were performed to observe ONFH. Apoptosis was assessed by TdT-mediated dUTP nick end labeling (TUNEL). Immunohistochemical staining was carried out to detect OCN and COL1. RESULTS: Ethanol significantly suppressed ALP activity, decreased gene expression of OCN and BMP2, lowered levels of RUNX2 protein, and reduced immunofluorescence staining of OCN and COL1 and calcium formation of hBMSCs. However, these inhibitory effects were attenuated by cordycepin co-treatment at concentrations of 1 and 10 µg/mL Moreover, it was revealed that the osteo-protective effect of cordycepin was associated with modulation of the Wnt/ß-catenin pathway. In vivo, by micro-CT, TUNEL and immunohistochemical staining of OCN and COL1, we found that cordycepin administration prevented alcohol-induced ONFH. CONCLUSION: Cordycepin treatment to enhance osteogenesis may be considered a potential therapeutic approach to prevent the development of alcohol-induced ONFH.

Relationships between tissue composition and viscoelastic properties in human trabecular bone.

Trabecular bone is a metabolically active tissue with a high surface to volume ratio. It exhibits viscoelastic properties that may change during aging. Changes in bone properties due to altered metabolism are sensitively revealed in trabecular bone. However, the relationships between material composition and viscoelastic properties of bone, and their changes during aging have not yet been elucidated. In this study, trabecular bone samples from the femoral neck of male cadavers (n=21) aged 17-82 years were collected and the tissue level composition and its associations with the tissue viscoelastic properties were evaluated by using Raman microspectroscopy and nanoindentation, respectively. For composition, collagen content, mineralization, carbonate substitution and mineral crystallinity were evaluated. The calculated mechanical properties included reduced modulus (Er), hardness (H) and the creep parameters (E1, E2, η1and η2), as obtained by fitting the experimental data to the Burgers model. The results indicated that the creep parameters, E1, E2, η1and η2, were linearly correlated with mineral crystallinity (r=0.769-0.924, p<0.001). Creep time constant (η2/E2) tended to increase with crystallinity (r=0.422, p=0.057). With age, the mineralization decreased (r=-0.587, p=0.005) while the carbonate substitution increased (r=0.728, p<0.001). Age showed no significant associations with nanoindentation parameters. The present findings suggest that, at the tissue-level, the viscoelastic properties of trabecular bone are related to the changes in characteristics of bone mineral. This association may be independent of human age.

Periosteal remodeling at the femoral neck in nonhuman primates.

UNLABELLED: Periosteal bone turnover is poorly understood. We documented intramembranous periosteal bone turnover in the femoral neck in intact nonhuman primates and an increase in osteoclast numbers at the periosteal surface in sex steroid-deficient animals. Our studies are the first to systematically document periosteal turnover at the femoral neck. INTRODUCTION: Bone size is an important determinant of bone strength, and cellular events at the periosteal surface could alter bone dimensions. We characterized periosteal cellular activity with dynamic histomorphometric studies of nonhuman primate femoral neck and shaft. MATERIALS AND METHODS: Femur specimens from 16 intact adult male and female nonhuman primates (Rhesus [Macaca mulatta, n = 9] and Japanese Macaque [Macaca fuscata, n = 7]) were analyzed. Animals were double-labeled with tetracycline, and necropsy was performed 2-7 days after the last dose. We characterized periosteal resorptive activity in an additional group of five intact and four castrate female animals. Multiple group comparisons in intact animals were performed by one-way ANOVA followed by a Fisher PLSD posthoc test. In gonadectomized animals, Fisher's exact test was used for dichotomous and Mann-Whitney U-test for continuous variables. RESULTS: Bone turnover in the periosteum of the femoral neck in intact animals was more rapid than at the femoral shaft but slower than in femoral neck cancellous bone. Similarly, in these intact animals, the eroded surface of cortical bone at the femoral neck periosteal surface was significantly greater than in the cancellous bone compartment (p < 0.0001) or on the femoral shaft (p < 0.0001). Gonadectomized female animals showed an increase in osteoclast number on the periosteal surface compared with intact controls (p < 0.01). CONCLUSIONS: We documented intramembranous periosteal bone turnover in the femoral neck by histomorphometric analyses. The tissue level bone formation rate was sufficient to add substantively to femoral neck size over time. Periosteal osteoclastic activity was not the result of the emergence of intracortical tunneling at the bone surface. Sex steroid deficiency produced an increase in osteoclast numbers at the periosteal surface. This is the first systematic documentation of periosteal turnover at the femoral neck.

Human femoral neck has less cellular periosteum, and more mineralized periosteum, than femoral diaphyseal bone.

Periosteal expansion enhances bone strength and is controlled by osteogenic cells of the periosteum. The extent of cellular periosteum at the human femoral neck, a clinically relevant site, is unclear. This study was designed to histologically evaluate the human femoral neck periosteal surface. Femoral neck samples from 11 male and female cadavers (ages 34-88) were histologically assessed and four periosteal surface classifications (cellular periosteum, mineralizing periosteum, cartilage, and mineralizing cartilage) were quantified. Femoral mid-diaphysis samples from the same cadavers were used as within-specimen controls. The femoral neck surface had significantly less (P<0.05) cellular periosteum (18.4+/-9.7%) compared to the femoral diaphysis (59.2+/-13.8%). A significant amount of the femoral neck surface was covered by mineralizing periosteal tissue (20-70%). These data may provide an alternate explanation for the apparent femoral neck periosteal expansion with age and suggest the efficiency of interventions that stimulate periosteal expansion may be reduced, albeit still possible, at the femoral neck of humans.

NOS isoforms in adult human osteocytes: multiple pathways of NO regulation?

Until now, eNOS has been considered to be the predominant osteocytic nitric oxide synthase (NOS) isoform in bone. We previously studied the distribution of eNOS protein expression in the human femoral neck because of its possible involvement in the response to load. Studies in rat and human fracture callus have shown that nNOS mRNA is expressed sometime after fracture, but no study has yet immunolocalized NOS isoforms in mature adult human bone. In this study, we have examined the distribution of NOS isoforms in iliac osteocytes. Frozen sections (10 microm) were cut from transiliac biopsies from 8 female osteoporotic patients (range, 56-80 years) and from 7 female postmortem femoral neck biopsies (range, 65-90 years). Sections were incubated overnight in antiserum for eNOS, nNOS, or iNOS followed by peroxidase/VIP substrate detection. We used eNOS and iNOS antisera directed against the C-terminus. For nNOS, three different antisera were used, two binding to different C-terminal epitopes and one binding to N-terminal epitope. Sections were then incubated in propidium iodide or methyl green to detect all osteocytes. eNOS antibody was able to detect eNOS epitopes in osteocytes. All three nNOS antibodies detected nNOS epitopes in osteocytes, but those directed against the C-terminus had higher detection rates. iNOS was rarely seen. In the iliac crest, the percentage of osteocytes positive for nNOS was higher than that for eNOS (cortical: nNOS 84.04%, eNOS 61.78%, P < 0.05; cancellous: nNOS 82.33%, eNOS 65.21%, P < 0.05). In the femoral neck, the percentage of osteocytes positive for nNOS (60.98%) was also higher than that for eNOS (40.41%), although this difference was not statistically significant. In conclusion, both eNOS and nNOS isoforms are present in osteocytes in the iliac crest and femoral neck.

Increased femoral neck cancellous bone and connectivity in coxarthrosis (hip osteoarthritis).

Patients with coxarthrosis (cOA) have a reduced incidence of intracapsular femoral neck fracture, suggesting that cOA offers protection. The distribution of bone in the femoral neck was compared in cases of coxarthrosis and postmortem controls to assess the possibility that disease-associated changes might contribute to reduced fragility. Whole cross-section femoral neck biopsies were obtained from 17 patients with cOA and 22 age- and sex-matched cadaveric controls. Densitometry was performed using peripheral quantitated computed tomography (pQCT) and histomorphometry on 10-microm plastic-embedded sections. Cortical bone mass was not different between cases and controls (P > 0.23), but cancellous bone mass was increased by 75% in cOA (P = 0.014) and histomorphometric cancellous bone area by 71% (P < 0.0001). This was principally the result of an increase of apparent density (mass/vol) of cancellous bone (+45%, P = 0.001). Whereas cortical porosity was increased in the cases (P < 0.0001), trabecular width was also increased overall in the cases by 52% (P < 0.001), as was cancellous connectivity measured by strut analysis (P < 0.01). Where osteophytic bone was present (n = 9) there was a positive relationship between the amount of osteophyte and the percentage of cancellous area (P < 0.05). Since cancellous bone buttresses and stiffens the cortex so reducing the risk of buckling, the increased cancellous bone mass and connectivity seen in cases of cOA probably explain, at least in part, the ability of patients with cOA to resist intracapsular fracture of the femoral neck during a fall.

Collagen fiber orientation in the femoral necks of apes and humans: do their histological structures reflect differences in locomotor loading?

Human hip joint anatomy differs substantially from that in other primates. Humans modulate pelvic tilt during walking with a highly developed abductor apparatus, but other primates (such as chimpanzees) clearly lack such an apparatus (they exhibit a Trendelenburg gait during bipedal progression). Because the primate femoral neck is cantilevered whenever it supports body mass, it must be consistently subjected to substantial bending at the neck/shaft interface during stance phase in quadrupedal or bipedal locomotion. It has been argued, however, that the powerful abduction during the single support phase in humans results in almost entirely compressive stress on the human femoral neck. We examined collagen fiber orientation in human and chimpanzee femoral neck cortices using circularly polarized light, which has been shown to be a strong correlate of bone loading patterns. Chimpanzee superior femoral neck cortex was shown to be largely nonbirefringent (dark), but the inferior cortex was strongly birefringent. Human femoral necks showed strong birefringence both superiorly and inferiorly. These results are consistent with loading patterns suggested from anatomical structure, and provide corroborative evidence of bone's ability to preferentially orient collagen fibers during extracellular matrix deposition.

Patterns of osteocytic endothelial nitric oxide synthase expression in the femoral neck cortex: differences between cases of intracapsular hip fracture and controls.

Evidence indicates that extensive amalgamation of adjacent resorbing osteons is responsible for destroying the microstructural integrity of the femoral neck's inferior cortex in osteoporotic hip fracture. Such osteonal amalgamation is likely to involve a failure to limit excessive resorption, but its mechanistic basis remains enigmatic. Nitric oxide (NO) inhibits osteoclastic bone destruction, and in normal bone cells its generation by endothelial nitric oxide synthase (eNOS, the predominant bone isoform) is enhanced by mechanical stimuli and estrogen, which both protect against fracture. To determine whether eNOS expression in osteocytes reflects their proposed role in regulating remodeling, we have examined patterns of osteocyte eNOS immunolabeling in the femoral neck cortex of seven cases of hip fracture and seven controls (females aged 68-96 years). The density of eNOS+ cells (mm(-2)) was 53% lower in the inferior cortex of the fracture cases (p < 0.0004), but was similar in the superior cortex. eNOS+ osteocytes were, on average, 22% further from their nearest blood supply, than osteocytes in general (p < 0.0001) and the nearest eNOS+ osteocyte was 57% further from its nearest canal surface (p < 0.0001). This differential distribution of eNOS+ osteocytes was significantly more pronounced in the cortices of fracture cases (p < 0.0001). We conclude that the normal regional and osteonal pattern of eNOS expression by osteocytes is disrupted in hip fracture, particularly at sites that are loaded most by physical activity. These results suggest that eNOS+ osteocytes may normally act as sentinels confining resorption within single osteons. A reduction in their number, coupled to an increase in their remoteness from canal surfaces, may thus permit the irreversible merging of resorbing osteons, and thus contribute to the marked increase in the fragility of osteoporotic bone.

Osteocyte lacunar occupancy in the femoral neck cortex: an association with cortical remodeling in hip fracture cases and controls.

In adult humans, osteocytes die and disappear from their lacunae in the cortex of bones which remodel slowly, such as the proximal femur, and osteocyte death is particularly prevalent in the elderly. We have investigated the statistical determinants of osteocyte density in microscopic fields (0.71 mm2) within thin, complete femoral neck cross-sections cut from biopsies embedded in methyl methacrylate and stained with solochrome cyanine R. Lacunae were counted under phase contrast and osteocytes within lacunae were counted in the same fields under epifluorescence. The percentage of lacunae containing an osteocyte varied between 12.4% and 99.2%, according to subject and quadrantic region of the cortex examined. The microscopic determinants of field-specific osteocyte density included the porosity measured in the field itself and the regional measurement of the proportion of cortical canals bearing osteoid. There was significant variation between subjects and, within subjects, between cortical regions. Also the inferior region showed a significantly higher density of lacunae than the superior region (+8.2%; P = 0.013). However, cases of fracture were not significantly different from controls with respect to osteocyte lacunar occupancy after adjusting for osteoid-bearing canals and porosity. It is concluded that in subjects in their 7th-9th decades of age, osteocyte lacunar occupancy is statistically associated with bone turnover, implying that high turnover (locally young bone age) might favor lacunar occupancy (ln% osteoid; P = 0.021). Alternative explanations of the association are that porosity reflects a better nutritional supply via the vasculature or that porosity of the cortex is associated with osteocyte density through an effect of osteocytes on bone remodeling.

Bone resorption at the femoral neck is dependent on local factors in nonosteoporotic late postmenopausal women: an in vitro-in vivo study.

Local mediators of bone resorption may be involved in bone loss in recently postmenopausal women and in osteoporosis. In the present study, we investigated the production of cytokines and the formation of osteoclast-like cells in marrow cultures from 16 late postmenopausal nonosteoporotic women (mean age: 66 +/- 8 years; time after menopause: 15 +/- 8 years) undergoing hip replacement for arthrosis. Marrow adherent mononuclear cells (MMNC) isolated from femoral diaphysis marrow were cultured for 10 days in the absence or in the presence of 1,25(OH)2D3. In vivo bone resorption was concomitantly assessed by histomorphometry on femoral neck bone sections. The number of TRAP+ multinucleated cells obtained after 10 days in MMNC cultured in the presence of 1,25(OH)2D3 correlated with the number of osteoclasts measured on the bone femoral neck biopsies (r = 0.65, p < 0.01), suggesting that the formation of multinucleated cells in vitro could reflect the osteoclast differentiation in vivo. Furthermore, the number of osteoclasts was related to the eroded volume and the trabecular separation of the femoral neck bone biopsies. Finally, the release of interleukin-1 (IL-1), IL-6, and TNF-alpha by cultures of peripheral blood mononuclear cells (PBMC) and MMNC was measured by radioimmunoassay. The cytokine levels of basal and 1,25(OH)2D3-treated MMNC decreased from days 2 to 5 and then reached a plateau to day 10. The number of TRAP+ multinucleated cells obtained after 10 days in MMNC cultures correlated with the basal IL-6 release in the same cultures determined at day 2 (r = 0.55, p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Primary cultures of human bone-derived cells produce parathyroid hormone-related protein: a study of 40 patients of varying age and pathology.

Parathyroid hormone-related protein (PTHrP), a mediator of hypercalcemia of malignancy, has been detected in many tumours and in some normal foetal and adult tissues. PTHrP has potent effects on bone turnover in vivo and in vitro. In this study we cultured cells derived from explants of bone obtained from 40 subjects (age range 2-88 years). Immunoreactive PTHrP (iPTHrP) was detected by immunoradiometric assay (IRMA) in conditioned medium from 25 of 40 cultures of bone-derived cells. PTHrP mRNA was detected in bone-derived cells by reverse transcriptase-linked polymerase chain reaction (RT-PCR). The identity of PCR products was confirmed by Southern blotting. Local production of PTHrP in vivo may be important in the regulation of bone growth and remodelling.