Gut Microbiota, Immune System, and Bone.

The gut microbiota (GM) is the whole of commensal, symbiotic, and pathogenic microorganisms living in our intestine. The GM-host interactions contribute to the maturation of the host immune system, modulating its systemic response. It is well documented that GM can interact with non-enteral cells such as immune cells, dendritic cells, and hepatocytes, producing molecules such as short-chain fatty acids, indole derivatives, polyamines, and secondary bile acid. The receptors for some of these molecules are expressed on immune cells, and modulate the differentiation of T effector and regulatory cells: this is the reason why dysbiosis is correlated with several autoimmune, metabolic, and neurodegenerative diseases. Due to the close interplay between immune and bone cells, GM has a central role in maintaining bone health and influences bone turnover and density. GM can improve bone health also increasing calcium absorption and modulating the production of gut serotonin, a molecule that interacts with bone cells and has been suggested to act as a bone mass regulator. Thus, GM manipulation by consumption of antibiotics, changes in dietary habits, and the use of pre- and probiotics may affect bone health. This review summarizes evidences on the influence of GM on immune system and on bone turnover and density and how GM manipulation may influence bone health.

Treatment with intermittent PTH increases Wnt10b production by T cells in osteoporotic patients.

UNLABELLED: We evaluated the effect of parathyroid hormone (PTH) on Wnt10b production by immune system cells in humans. We showed that bone anabolic effect of intermittent PTH treatment may be amplified by T cells through increased production of Wnt10b. Chronic increase in PTH as in primary hyperparathyroidism does not increase Wnt10b expression. INTRODUCTION: The aim of this study is to assess the effect of PTH on Wnt10b production by immune system cells in humans. We assessed both the effect of intermittent PTH administration (iPTH) and of chronic PTH hypersecretion in primary hyperparathyroidism (PHP). METHODS: Eighty-two women affected by post-menopausal osteoporosis were randomly assigned to treatment with calcium and vitamin D alone (22) or plus 1-84 PTH (42), or intravenous ibandronate (18). Wnt10b production by unfractioned blood nucleated cells and by T, B cells and monocytes was assessed by real-time RT-PCR and ELISA at baseline, 3, 6, 12 and 18 months of treatment. The effect of chronic elevation of PTH was evaluated in 20 patients affected by PHP at diagnosis and after surgical removal of parathyroid adenoma. WNT10b from both osteoporotic and PHP patients was compared to healthy subjects matched for age and sex. RESULTS: iPTH increases Wnt10b production by T cells, whereas PHP does not. After surgical restoration of normal parathyroid function, WNT10b decreases, although it is still comparable with healthy subjects' level. Thus, chronic elevation of PTH does not significantly increase WNT10b production as respect to control. CONCLUSIONS: This is the first work showing the effect of both intermittent and chronic PTH increase on Wnt10b production by immune system cells. We suggest that, in humans, T cells amplified the anabolic effect of PTH on bone, by increasing Wnt10b production, which stimulates osteoblast activity.

Improving adherence to and persistence with oral therapy of osteoporosis.

UNLABELLED: Osteoporosis treatment has low adherence and persistence. This study evaluated if greater patient involvement could improve them. At 12 months, only 114 out of 344 participants were "fully adherent and persistent" (all drug doses taken throughout the study). Only frequency of drug administration had a significant influence on adherence. INTRODUCTION: Osteoporosis affects millions of individuals worldwide. There are now several effective drugs, but adherence to and persistence with treatment are low. This 12-month multicenter, prospective, randomized study evaluated the efficacy of two different methods aimed at improving adherence and persistence through greater patient involvement, compared with standard clinical practice. METHODS: Three hundred thirty-four post-menopausal women, receiving an oral prescription for osteoporosis for the first time, were recruited and randomized into three groups: group 1 (controls, managed according to standard clinical practice) and groups 2 and 3 (managed with greater patient and caregiver involvement and special reinforcements: group 2, instructed to use several different "reminders"; group 3, same "reminders" as group 2, plus regular phone calls from and meetings at the referring Center). All enrolled women had two visits (baseline and 12 months). RESULTS: Of 334 enrolled women, 247 (74%) started the prescribed therapy. Of those who started, 219 (88.7%) persisted in therapy for at least 10 months. At final evaluation, only 114 women were considered as "fully adherent and persistent" (all doses taken throughout the 12 months). There were no significant differences regarding "full adherence" among the three randomized groups. The frequency of drug administration had a significant influence: weekly administration had a >5-fold higher adherence and monthly administration an 8-fold higher adherence (p < 0.0001) than daily administration. CONCLUSIONS: The special effort of devising and providing additional reminders did not prove effective. Additional interventions during the follow-up, including costly interventions such as phone calls and educational meetings, did not provide significant advantages.

Primary breast cancer stem-like cells metastasise to bone, switch phenotype and acquire a bone tropism signature.

BACKGROUND: Bone metastases represent a common and severe complication in breast cancer, and the involvement of cancer stem cells (CSCs) in the promotion of bone metastasis is currently under discussion. Here, we used a human-in-mice model to study bone metastasis formation due to primary breast CSCs-like colonisation. METHODS: Primary CD44⁺CD24⁻ breast CSCs-like were transduced by a luciferase-lentiviral vector and injected through subcutaneous and intracardiac (IC) routes in non-obese/severe-combined immunodeficient (NOD/SCID) mice carrying subcutaneous human bone implants. The CSCs-like localisation was monitored by in vivo luciferase imaging. Bone metastatic CSCs-like were analysed through immunohistochemistry and flow cytometry, and gene expression analyses were performed by microarray techniques. RESULTS: Breast CSCs-like colonised the human-implanted bone, resulting in bone remodelling. Bone metastatic lesions were histologically apparent by tumour cell expression of epithelial markers and vimentin. The bone-isolated CSCs-like were CD44⁻CD24⁺ and showed tumorigenic abilities after injection in secondary mice. CD44⁻CD24⁺ CSCs-like displayed a distinct bone tropism signature that was enriched in genes that discriminate bone metastases of breast cancer from metastases at other organs. CONCLUSION: Breast CSCs-like promote bone metastasis and display a CSCs-like bone tropism signature. This signature has clinical prognostic relevance, because it efficiently discriminates osteotropic breast cancers from tumour metastases at other sites.

Platelet vitamin D receptor is reduced in osteoporotic patients.

AIM: It is well known that vitamin D plays an important role in maintaining bone homeostasis and in regulating calcium absorption. The active form of vitamin D interacts with its receptor the VDR that is expressed in multiple tissues and it is involved in platelets (PLTs) function. In the present study we evaluate PLTs' VDR expression in osteoporotic as opposed to healthy subjects. METHODS: We enrolled in the study 77 women with postmenopausal osteoporosis, 33 healthy women of childbearing age, 49 healthy men, and 11 healthy women matched with patients for age and postmenopausal period. Thirty-nine patients had had one femoral fracture occurred after the age of fifty and attributable to primary osteoporosis. Bone mineral density, markers of bone metabolism and VDR levels were measured in all the subjects. RESULTS: Our data show that VDR level is lower in patients as respect to controls and is positively correlated with bone density, but not with markers of bone metabolism. We also found a decrease in the phosphorus levels in patients without differences in vitamin D levels and in the dietary calcium intake. CONCLUSION: The lower VDR expression in osteoporotic could indicate a lower ability to respond to vitamin D, and could be the explanation of the increase in the PTH and decrease in the phosphorus levels in patients with respect to controls.

Teriparatide increases the maturation of circulating osteoblast precursors.

UNLABELLED: This study shows that teriparatide promotes the circulating osteoblast (OB) precursor degree of maturation in patients affected by postmenopausal osteoporosis. INTRODUCTION: Anabolic treatment with teriparatide has proven effective for the therapy of postmenopausal osteoporosis and significantly reduces the risk of non-vertebral fragility fractures. The aim of this study was to investigate the effect of teriparatide on circulating OB precursors. METHODS: We evaluated by flow cytometry and real-time PCR the expression of OBs typical markers in peripheral blood mononuclear cells during treatment with teriparatide plus calcium and vitamin D, raloxifene plus calcium and vitamin D or calcium and vitamin D alone at various time points. Serum bone alkaline phosphatase and osteocalcin (OC) were measured as markers of bone turnover. RESULTS: Our results show that circulating OB precursors are more numerous and more immature in patients affected by fragility fractures than in osteoporotic patients without fractures. We also show that teriparatide treatment increases the expression of alkaline phosphatase and of OC in OB precursors; thus, it increases their degree of maturation. CONCLUSIONS: We suggest that teriparatide acts as anabolic agents also by promoting the maturation of OB precursors.

Clinical characteristics and incidence of first fracture in a consecutive sample of post-menopausal women attending osteoporosis centers: The PROTEO-1 study.

BACKGROUND: Osteoporosis is a highly prevalent disease and fractures are a major cause of disability and morbidity. AIM: The purpose of this study was to characterize post-menopausal women attending osteoporosis centers in Italy, to evaluate physician management, and to determine the incidence of first osteoporotic fracture. SUBJECTS AND METHODS: PROTEO-1 was an observational longitudinal study with a 12-month follow-up. Data were collected from women attending osteoporosis centers. Women without prevalent fracture were eligible to enter the 1-yr follow-up phase: the clinical approach to patients according to their fracture risk profile and the incidence of fracture were recorded. RESULTS: 4269 patients were enrolled in 80 centers in the cross-sectional phase; 34.2% had an osteoporotic fracture at baseline. Patients with prevalent fractures were older and more likely to be treated compared with non-fractured patients. The incidence of vertebral or hip fracture after 1 yr was 3.84%, regardless of the calculated risk factor profile, and was significantly higher in patients with back pain at baseline (4.2%) compared with those without back pain (2.2%; p=0.023). Generally, physicians prescribed more blood exams and drugs to patients at higher risk of fracture. Among fractured patients only 24% were properly treated; the rate of non-responders to treatment was about 4%. CONCLUSIONS: In a large, unselected sample of post-menopausal women attending osteoporosis centers, those without previous fracture were at substantial risk of future fracture, regardless of their theoretical low 10-yr fracture risk. The presence of back pain in women without previous fracture warrants close attention.

Bone and bone marrow pro-osteoclastogenic cytokines are up-regulated in osteoporosis fragility fractures.

UNLABELLED: This study evaluates cytokines production in bone and bone marrow of patients with an osteoporotic fracture or with osteoarthritis by real time PCR, Western blot and immunohistochemistry. We demonstrate that the cytokine pattern is shifted towards osteoclast activation and osteoblast inhibition in patients with osteoporotic fractures. INTRODUCTION: Fragility fractures are the resultant of low bone mass and poor bone architecture typical of osteoporosis. Cytokines involved in the control of bone cell maturation and function are produced by both bone itself and bone marrow cells, but the roles of these two sources in its control and the amounts they produce are not clear. This study compares their production in patients with an osteoporotic fracture and those with osteoarthritis. METHODS: We evaluated 52 femoral heads from women subjected to hip-joint replacement surgery for femoral neck fractures due to low-energy trauma (37), or for osteoarthritis (15). Total RNA was extracted from both bone and bone marrow, and quantitative PCR was used to identify the receptor activator of nuclear factor kB Ligand (RANKL), osteoprotegerin (OPG), macrophage colony stimulating factor (M-CSF), transforming growth factor ß (TGFß), Dickoppf-1 (DKK-1) and sclerostin (SOST) expression. Immunohistochemistry and Western blot were performed in order to quantify and localize in bone and bone marrow the cytokines. RESULTS: We found an increase of RANKL/OPG ratio, M-CSF, SOST and DKK-1 in fractured patients, whereas TGFß was increased in osteoarthritic bone. Bone marrow produced greater amounts of RANKL, M-CSF and TGFß compared to bone, whereas the production of DKK-1 and SOST was higher in bone. CONCLUSIONS: We show that bone marrow cells produced the greater amount of pro-osteoclastogenic cytokines, whereas bone cells produced higher amount of osteoblast inhibitors in patients with fragility fracture, thus the cytokine pattern is shifted towards osteoclast activation and osteoblast inhibition in these patients.

Alendronate reduces osteoclast precursors in osteoporosis.

UNLABELLED: This study evaluates the effect of alendronate on osteoclastogenesis, cytokine production, and bone resorption in postmenopausal women. We suggest that it acts on mature bone resorbing osteoclasts after 3 months of treatment, whereas, after 1 year, it diminishes their formation by reducing their precursors and serum RANKL. INTRODUCTION: Osteoclasts are the target cells of bisphosphonates, though the most drug-sensitive steps of their formation and activity have not been determined. The present study evaluates the effect of alendronate on osteoclastogenesis, cytokine production, and bone resorption in postmenopausal women. METHODS: The study was conducted on 35 osteoporotic women; 15 were pretreated with alendronate 70 mg/week, whereas, 20 were treated with calcium 1 g/day and vitamin D 800 IU/day. After 3 months, 30 received alendonate 70/mg, vitamin D 2800 IU/week, and calcium 1 g/day for 12 months (combined therapy), whereas, the other five patients remained on calcium 1 g/day and vitamin D 800 IU/day. The following parameters were assessed before and after therapy: changes in bone resorption markers, circulating osteoclast precursors, formation of osteoclasts in peripheral blood mononuclear cell cultures, their viability, and variations in cytokines production. RESULTS: After 3 months of alendronate, there was no significant reduction in the number of osteoclast precursors, osteoclast formation and viability, and cytokine levels, whereas, there was a significant reduction of bone resorption markers. One year of the combined therapy, on the other hand, reduced osteoclast precursors, osteoclast formation, and serum RANKL, whereas, calcium plus vitamin D alone had no effect. CONCLUSIONS: We suggest that alendronate mainly acts on mature bone resorbing osteoclasts in the short term, whereas, its long-term administration diminishes their formation by reducing their precursors and serum RANKL.

The osteoprotegerin/RANK/RANKL system: a bone key to vascular disease.

Osteoporosis and atherosclerosis are degenerative disorders of old age that often present together, but recently it has been suggested that the association between osteoporosis and cardio-vascular diseases is not just due to the aging process. The osteoprotegerin (OPG)/receptor activator of nuclear factor-kB (RANK)/RANK ligand (RANKL) system has been identified as a possible mediator of arterial calcification suggesting common links between osteoporosis and vascular diseases. Since the discovery of the OPG/RANK/RANKL system, much has been learned about its role in controlling skeletal biology; however, its role in the context of vascular biology is only beginning to be explored. It has been suggested that OPG might act as an autocrine/paracrine regulator of vascular calcification and might be useful as a serum marker of vascular disease. However, the exact role of OPG (or RANKL/RANK) in vascular calcification is still not completely understood. This review aims to report the recent findings on the relationship between osteoporosis and OPG/RANK/RANKL-mediated vascular disease.

HDL cholesterol and bone mineral density in normal-weight postmenopausal women: is there any possible association?

AIM: Epidemiological investigation of the association between lipid profile, atherosclerosis and bone mass has produced conflicting RESULTS: The present paper reports the assessment of the lipid profile, bone mineral density (BMD) and turnover in a cohort of Italian women. METHODS: In this cross sectional study we enrolled 173 women in menopause (101 osteoporotic and 72 normal). In each subject the authors evaluated BMD, bone turnover, lipid profile (total cholesterol, high density lipoprotein [HDL], low density lipoprotein [LDL] and triglycerides), and risk factors for osteoporosis, cardiovascular diseases and eating habits using a questionnaire. RESULTS: HDL was significantly higher in osteoporotic patients than in controls and the risk of osteoporosis was significantly higher in women with higher level of HDL. The authors suggest that the level of HDL could be used as screening for postmenopausal osteoporosis: the cut-off points recommended are HDL >61 mg/dL to detect women with a high risk (sensitivity 74%) and <45 mg/dL to detect those with a low risk (specificity 83%). CONCLUSION: This study provides evidences of the relation between HDL, but not total cholesterol or LDL levels with BMD in a cohort of normal-weight women and equally distributed cardiovascular risks. It also suggests that a proatherogenic lipid profile is associated with higher bone mineral density, and that HDL can be used in deciding whether a patient's BMD should be measured.

Protein intake: the impact on calcium and bone homeostasis.

Bone density depends on various factors such as age, hormonal status, genetics factors and lifestyle: a balanced diet plays a fundamental role in the prevention of osteoporosis. The role of protein intake on bone health is still controversial: this review is focused on the relation between protein intake and bone metabolism.

Hypovitaminosis D in internal medicine inpatients.

Some studies have suggested that hypovitaminosis D may be a consequence of protein-calorie malnutrition. This study assessed both the relationship between vitamin D status, malnutrition, calcium and phosphorus metabolism indices and the importance attached by internists to these alterations. There were 239 patients admitted to an internal medicine division who underwent examinations to assess nutritional state, liver and renal function, and bone metabolism. At the end of the study, the clinical data included in the discharge letter, the treatment prescribed, and the diagnosis assigned to patients on their hospital discharge form were collected. Hypovitaminosis D was found in 72% and hypoalbuminemia in 34.3% of patients. Subjects with hypovitaminosis were generally older and had lower albumin levels than those with mild or no hypovitaminosis. 25-Hydroxyvitamin D was inversely related with parathyroid hormone and directly related with albumin. Alterations of calcium and phosphorus metabolism were present in 55.6% and recorded by the division's physicians for only 13.53% of patients, of whom 72.37% were not specifically treated. There is a direct correlation between 25-hydroxyvitamin D and albumin levels. The high incidence and the metabolic consequence of hypovitaminosis D and of protein-calorie malnutrition is significantly underestimated and undertreated by physicians.

Effects of lifestyle and risk factors on bone mineral density in a cohort of Italian women: suggestion for a new decision rule.

In this study the authors analyzed the role of risk factors in postmenopausal osteoporosis in a cohort of Italian women and evaluated predictive values of decision rules for early identification of osteoporotic women. Furthermore, the authors investigated the prevalence of secondary osteoporosis in this population. Women who underwent bone densitometry were asked to answer a questionnaire about the common risk factors for osteoporosis. Patients were classified as nonosteoporotic, nonosteopenic, and osteoporotic. Risk factors were compared among the groups by use of analysis of variance (ANOVA). National Osteoporosis Foundation (NOF) recommendation, Osteoporosis Risk Assessment Instruments (ORAIs), Osteoporosis Self-Assessment Tools (OST) score, and weight criterion were applied to this population. The authors proposed a new decision rule based on a new score. A total of 525 women received the questionnaire: 47.4% women were osteoporotic, 32.2% were osteopenic, and 20.4% nonosteoporotic. Risk factors that differed significantly between these groups were: age, age at menarche, postmenopausal period, and body mass index (BMI); the aforementioned risk factors appear to be significant predictors of bone density (BMD) in linear regression model. The incidence of secondary osteoporosis was 13%. In conclusion, the authors (1) confirmed the role played by nonmodifiable risk factors in determining BMD; (2) showed that the use of NOF guidelines, ORAI, OST score, and weight criterion is not satisfactory in our cohort; (3) suggested a new score, based upon the features that were significantly different between patients and controls; and (4) demonstrated the relatively high prevalence of secondary osteoporosis and suggest a primary screening for secondary osteoporosis in all patients with low BMD.

Is leptin the link between fat and bone mass?

Leptin is a cytokine-like hormone which is considered the link between fat and bone; it is produced by adipocytes and osteoblasts, regulates food intake via specific receptors located in the central nervous system (CNS) and bone mass through alternate pathways: one involving a direct stimulatory effect on bone formation; and another indirect effect through the CNS that suppresses bone formation. Leptin exerts a direct stimulatory effect on osteoblast differentiation and on bone growth if directly administered, while it exerts an inhibitory effect on bone formation if administered in the CNS. It is therefore unclear whether leptin should be considered an antiosteogenic factor or an anabolic agent for bone formation: in all probability, leptin has a broader role in human physiology and in particular its action has evolved in order to synchronize periods of bone growth, mineral accretion and fertility with periods of food availability, while it restricts growth and reproduction during periods of nutritional stress.

Effects of potassium citrate supplementation on bone metabolism.

Western diets rich in animal protein result in long-term acid loading that, despite corresponding increases in net renal acid excretion, may induce a chronic state of acidemia. This may have deleterious effects on both the kidney and bone, by increasing the risk of calcium stone in the former and leading to chemical dissolution of mineral alkaline salts in the latter. Whereas supplementation with alkaline citrate has been shown to reduce stone recurrences, its effect on bone turnover has received less attention. The aim of the present study was to evaluate whether potassium citrate favorably affects bone turnover markers in postmenopausal females with low bone density. Thirty women, aged 58 +/- 8.1 years, were enrolled and studied on basal conditions and after a 3-month course of potassium citrate supplementation (0.08-0.1 g/kg b.w. daily). Twenty-two women concluded the study while 8 withdrew. Twenty-four age-matched healthy women were taken as control cases. All were evaluated for electrolyte and acid-base balance-related parameters, bone turnover, markers and renal function. A significant decrease in net acid excretion was observed upon citrate supplementation, and this was paralleled by a significant decrease of urinary deoxypyridinolines, hydroxyproline-to-creatinine ratios, and, to a lesser extent, serum osteocalcin. Percent variations of urine citrate were inversely related to those of deoxypyridinolines and hydroxyproline. No change in these chemistries occurred in the control group. Our results suggest that treatment with an alkaline salt, such as potassium citrate, can reduce bone resorption thereby contrasting the potential adverse effects caused by chronic acidemia of protein-rich diets.

Effect of raloxifene and clodronate on bone density in postmenopausal osteoporotic women.

The aim of the present study was to determine the safety and efficacy of combined therapy with raloxifene (RLX) and clodronate (CLD) in postmenopausal women. We enrolled 45 women with postmenopausal osteoporosis. The patients were randomly assigned to two different therapeutic groups: RLX 60 mg/day (n = 23) and RLX 60 mg/day plus CLD 100 mg intramuscularly (i.m.) once every 10 days (n = 22); 1 g of calcium and 800 IU of vitamin D3 were also given daily to both groups. Lumbar and femoral bone mineral density (BMD) were assessed at baseline and after 12 months of therapy using the dual X-ray absorptiometry technique (Norland XR36). We measured the bone turnover markers NTx and CTx, bone alkaline phosphatase (BAP) and osteocalcin at baseline and after 12 months of therapy. Our data demonstrate that 1 year of combined RLX+CLD therapy induced a higher increase in lumbar BMD than treatment with RLX alone as well as a major decrease in bone resorption markers, suggesting an additive effect of CLD on bone mass and inhibition of bone turnover. Furthermore, after 1 year of therapy levels of bone formation markers (osteocalcin and BAP) had increased in both groups, but the increase in osteocalcin and BAP was significantly higher in the RLX+CLD treated group, suggesting that, in addition to its inhibitory effects on resorption, CLD might also have stimulatory effects on mature osteoblast activity.

High density lipoproteins (HDL) in women with postmenopausal osteoporosis: a preliminary study.

OBJECTIVE: Since a previous study showed an inverse correlation between high density lipoproteins (HDL) and bone mineral density (BMD), we searched for a possible relationship between HDL level and the presence of postmenopausal osteoporosis. DESIGN: We measured HDL levels in 37 women with postmenopausal osteoporosis, and compared them with a control group of 43 healthy postmenopausal women. The HDL levels were compared between the two groups using Student's t test and were correlated with BMD by Pearson's coefficient. To avoid possible selection bias, we compared patients and controls for body mass index by chi 2 test. The sensitivity and specificity of HDL level higher than 65 mg% (positive test) or lower than 45 mg% (negative test) was compared with double emission x-ray absorptiometry (considered the gold standard in the measurement of BMD). RESULTS: The level of HDL was significantly higher in the osteoporotic patients than in the controls (67.7 +/- 15.5 mg% vs 58.3 +/- 11.6 mg%, p = 0.0039). HDL was inversely correlated with BMD (r = -0.29, p = 0.0083). HDL higher than 65 mg% has a high specificity (77%) for patients with osteoporosis, while HDL lower than 45 mg% has a high sensitivity (97%) in detecting subject without osteoporosis. CONCLUSIONS: Our preliminary data suggest an interesting, as yet unexplained association between HDL and bone mineral density in postmenopausal women.

Ossification centers of human femur.

The purpose of the present work was to investigate the appearance of ossification centers in human femur by Dual-energy X-ray Absorptiometry (DXA), comparing densitometric data with morphogenetic events. Posteroanterior scans were performed on 31 dried femora (from 11.5 weeks of conceptual age to 1 year of postnatal life) by a Hologic QDR 1000 X-ray densitometer with Ultra-Hi-Resolution software. The results were expressed as bone mineral content (BMC, g) and bone mineral density (BMD, g/cm2). The analyses were performed on a rectangular area corresponding to the minimum width and total length of the shaft. The rectangular area was divided into five equal sections along its longitudinal axis, and BMC was calculated on each section. To distinguish the ossification area of the lesser trochanter with respect to the area of the greater trochanter, the proximal femoral end was divided into two portions, medial and lateral, respectively; BMC and BMD were calculated on each portion. Our data show that the ossification center of the femoral shaft extends prevalently in the proximal direction. A denser area was recognizable on the densitometric images at the level of the lesser trochanter from the 19th week of prenatal life and at the level of the greater trochanter at term. During development, the trends of BMC and BMD are similar in both trochanteric areas, but these parameters are fairly constantly higher at the level of the lesser trochanter. Our findings disagree with the data of the current literature on the postnatal appearance of the trochanteric ossification centers and suggest a different biomechanical interpretation of the secondary ossification of the femur.