Iliac Crest Biopsies Research Articles

Side-to-side and within-side variability of 3D bone microarchitecture by conventional micro-computed tomography of paired iliac crest biopsies

Bone microarchitecture in osteoporosis can be characterized by examining iliac bone biopsies and treatment effects assessed by comparing a baseline biopsy from one side to a posttreatment biopsy from the other side, a method that assumes limited side-to-side variability. New techniques based on micro-computed tomography (µCT) provide information on the three-dimensional (3D) microarchitecture of bone. We used µCT to measure side-to-side and within-side variability of 3D microarchitectural parameters of trabecular and cortical bone in paired iliac-crest biopsies, one from each side. A Bordier needle trephine was used to collect biopsies from 30 postmenopausal female cadavers (mean age, 73.7±10.7 years; range, 55–96 years). Biopsies were chemically defatted then imaged using a desktop µCT scanner (voxel size, 10.77 µm). Parameters measured in trabecular bone consisted of bone volume/tissue volume (BV/TV, %), direct trabecular thickness and trabecular spacing (Tb.Th⁎ and Tb.Sp⁎, µm) using the sphere method, bone surface/bone volume (BS/BV, mm−1), trabecular number (Tb.N, mm−1), structure model index (SMI), trabecular pattern factor (Tb.Pf), and degree of anisotropy (DA). In cortical bone, we measured cortical thickness (Cort.Th), porosity (Cort.Porosity), and pore diameter (Po.Dm). For trabecular bone parameters, reproducibility as assessed from two µCT acquisitions ranged from 4.1% to 6.9%. To assess side-to-side variability, we matched the volumes of interest selected in the right and left iliac crests. The mean difference in absolute individual percent variation (mAbsΔind) between the two sides ranged from 10.8% to 14.8% for all trabecular parameters except Tb.Pf (74%) and SMI (84%). In cortical bone, mAbsΔind were 11.6% for Po.Dm, 15.1% for Cort.Porosity, and 27.6% for Cort.Th. To assess within-side variability, we divided the trabecular iliac crest volume into three equal parts, one adjacent to each cortex and one in the middle. Values of mAbsΔind versus the middle part were ranging from 7.6% for Tb.Sp⁎ to 26.2% for BV/TV. Thus, within-side variability was similar in magnitude to side-to-side variability. The considerable differences in robustness across trabecular parameters indicate a need for selecting the most stable parameters, most notably for longitudinal studies of small numbers of patients. Acquisition by µCT and image analysis must comply with stringent quality criteria, especially the distance from the cortices must be standardized.

Intratrabecular tunneling increases trabecular number throughout the skeleton of ovariectomized rhesus monkeys treated with parathyroid hormone 1–84

Daily treatment of ovariectomized (OVX) adult rhesus monkeys with human parathyroid hormone (PTH) 1–84 for 16 months increases trabecular bone volume (BV/TV), number (Tb.N) and connectivity at lumbar vertebra-3 (L3) and thoracic vertebra-10. We proposed that the increased Tb.N and connectivity was achieved by stimulation of intratrabecular tunneling. Using histomorphometry to determine frequency of events, we have now quantified intratrabecular tunneling at L3 and extended it to investigate the effects of PTH(1–84) treatment on trabecular bone at the proximal femur, distal radius and iliac crest of these animals. At L3, tunneling frequency was low in control sham and OVX animals (∼ 0.05/mm 2) but increased significantly in PTH(1–84)-treated animals (0.27, 0.49 and 0.95/mm 2 with the 5, 10 and 25 μg/kg doses, respectively). Very similar tunneling frequencies were observed at all skeletal sites in all groups. Iliac crest biopsies were also collected at baseline and after 6 months of treatment and showed significant time- and dose-related increases in tunnels. Although the pattern and magnitude of response varied slightly from site to site, PTH(1–84) treatment significantly increased Tb.N, as well as BV/TV and bone formation rate at all skeletal sites. A modest but statistically significant increase in trabecular thickness occurred only at the iliac crest. In summary, intratrabecular tunneling is rare in control monkeys, but increased substantially with PTH(1–84) treatment. This phenomenon provides a plausible explanation for the PTH(1–84)-induced increase in Tb.N observed in OVX monkeys. Moreover, these analyses allowed a comparison of the effects PTH(1–84) treatment on trabecular bone at multiple locations.

Hernie pariétale post-traumatique.Technique de réparation pariétale, à propos d’un cas

Little less than half of the occurrences acquired lumbar hernias are caused by traumatisms: direct parietal contusions, iliac crest biopsies or fractures. Regarding their frequency, they are rare but generally underdiagnosed. Abdominal wall reconstruction is motivated by the risk of hernia strangulation, but also aims to rebuild continent abdominal muscles, allowing the loss of discomfort or worsening risk as well as to resume physical activities. We report a case of parietal reconstruction of a traumatism-induced lumbar hernia in a 59-year-old male patient. Scanner showed lumbar disinsertion of abdominal transversus and both obliquus externus and internus muscles. The pressure exerted on abdominal muscles, greater than the elastic resistance of the insertion aponeurosis, caused their tearing. The flexibility and elasticity of the skin allowed the sustainment of its integrity. We applied Welti-Eudel's technique to suture the dorsal edge of the transverse and oblique intern muscles with a flap coming from lumbo-dorsal fasciae of sacrospinalis muscles. A parietal prosthesis is inserted between this deep level and the obliquus externus, which is restored. Fifteenth month's results, both morphological and functional, are excellent. Check scanner shows anatomical restitution of abdominal muscles. The scanner of abdominal muscles is the leading complementary exam. It is repeated with a gap, so that the hematoma does not disturb its interpretation. Surgical indication is definite for active adults. Parietal prosthetic strengthening, bone inserted between two muscular levels, avoid late loosening. It has no immediate mechanical value, which is secured by abdominal girdle during healing.

Bone disease after renal transplantation

Kidney transplantation largely restores defective exocrine and endocrine renal function in patients with end-stage renal disease (ESRD). This in turn is expected to lead to a progressive correction of established renal bone disease. This widely held assumption, however, lacks convincing evidence from experimental and clinical data published thus far. A major obstacle to the investigation of renal osteodystrophy in transplant recipients has been its unpredictable evolution under the multiple biochemical and hormonal influences that regulate mineral metabolism and bone turnover independently. The clinical and histological features of the pre-existing uraemic bone disease at the time of kidney transplantation are highly variable. Moreover, its course after transplantation depends on persisting abnormalities such as hypercalcaemia, hypophosphataemia and hypomagnesaemia as well as on the type, dose and duration of immunosuppressive medications that are needed to minimize allograft rejection. These factors operate additively and their effects are often difficult to dissociate from the already existing osteopathy. The existence and type of post-transplant bone disease may not be recognized correctly and appropriate therapy may not be started in time. Although glucocorticoid therapy represents a pathogenetic key factor other immunosuppressive drugs such as cyclosporine, tacrolimus, azathioprine and rapamycin clearly contribute to its prevalence and expression through their pleiotropic pharmacological effects. These drugs have been shown to increase overall bone turnover anduor to stimulate loss of bone mass independently. Based on currently available data, only mycophenolate mofetil appears to have a neutral effect in this regard [1]. Only a limited number of reports dealt with this problem in the past. There is an obvious need for welldesigned prospective studies with sufficient patient numbers to determine the relative importance of the numerous factors involved in the perturbed bone remodelling following kidney transplantation. Furthermore, a consensus-based classification of the various types of renal allograft-associated osteopathy, including clinical, radiological and histomorphological criteria, might lead to a more reliable basis for experimental research into this particular disease and facilitate the comparison between studies. When working up a patient for symptoms and signs of renal transplant-associated bone disease, clinical data for an effective evaluation and treatment of this condition are often lacking. A longitudinal analysis of the disease process would undoubtedly allow a more precise diagnosis and a more appropriate care of post-transplant bone disease. In many cases, follow-up data from the time of transplantation or before are unavailable or inaccessible. Radiographic examinations of the thoracic and lumbar spine with a bone densitometry of the femoral neck or other sites, and an iliac crest biopsy with a histomorphometric analysis in at last some patients should be ideally obtained before a diagnosis is made and a treatment is initiated. Several main osteological complications of kidney transplantation will be reviewed in the following, as summarized in Table 1.

The aminobisphosphonate risedronate preserves localized mineral and material properties of bone in the presence of glucocorticoids

Glucocorticoids (GCs) alter bone strength such that patients receiving these medications have a high rate of fragility-related fractures. The purpose of this study was to assess whether concurrent treatment with GCs (prednisolone) and risedronate (an aminobisphosphonate) would prevent the reduction in bone strength induced by GCs, in a mouse model of GC-induced bone loss and in patients enrolled in a clinical study. We evaluated mice treated with prednisolone pellets alone, GCs plus risedronate, or placebo alone and iliac crest biopsy specimens obtained from patients who were treated with GCs plus placebo or GCs plus risedronate for 1 year. We measured the mass, architecture, and physical and material properties of bone (subject to therapeutic treatments) at nanoscale to macroscopic dimensions, using synchrotron x-ray tomography, elastic modulus mapping, transmission electron microscopy, and small-angle x-ray scattering techniques. GC treatment reduced trabecular bone mass, microarchitecture, and the degree of bone mineralization and elastic modulus within the trabeculae. Concurrent treatment with GCs and risedronate prevented the deterioration of trabecular bone architecture, reduced the degree of mineralization, and preserved elastic modulus within the trabeculae, in both mouse and human bone. In addition, treatment with risedronate plus GCs in mice appeared to preserve bone crystal orientation, compared with treatment with GCs alone. Risedronate prevented the localized changes in mineral and material properties of bone induced by GCs, which may ultimately improve bone strength.

Human iliac crest cancellous bone elastic modulus and hardness differ with bone formation rate per bone surface but not by existence of prevalent vertebral fracture

The goals of this study were to measure tissue-level elastic moduli and hardness of human cancellous bone using nanoindentation, and determine the relationship between nanoindentation results and previously measured bone histomorphometric variables and bone mineralization. Forty iliac crest biopsies were used in this study, which were collected from Caucasian females with vertebral fracture or from a normal healthy female Caucasian population. They were also categorized into two groups according to high or low bone formation rate per bone surface (BFR/BS). Thirty-two sites were randomly selected on each specimen for nanoindentation with a Berkovich diamond indenter. Two sets of elastic moduli and hardness were calculated using the continuous stiffness measurement method and the Oliver-Pharr method, respectively. Relationships between nanoindentation results and donor age, bone mineralization, and histomorphometric variables were examined. No difference in elastic moduli or hardness was observed between the normal and fracture groups. Significantly lower elastic moduli were observed in the high BFR/BS group. The elastic moduli and hardness measurements were not significantly correlated with the bone mineralization measured independently in a previous study. Linear correlation between elastic modulus and hardness calculated using the Oliver-Pharr method was not different between the normal and fracture groups or between the high and low BFR/BS groups. Nanoindentation hardness was a very good predictor of bone tissue elastic modulus for both normal and osteoporotic bone tissues. Osteoporosis may not change the relationship between bone tissue elastic modulus, bone hardness, and bone mineralization.

Evidence that Treatment with Risedronate in Women with Postmenopausal Osteoporosis Affects Bone Mineralization and Bone Volume

Risedronate is used in osteoporosis treatment. Postmenopausal women enrolled in the Vertebral Efficacy with Risedronate Therapy trial received either risedronate (5 mg/day) or placebo for 3 years. Subjects received calcium and vitamin D supplementation if deficient at baseline. Lumbar spine bone mineral density (BMD) was measured at baseline and at 3 years. Quantitative back-scattered electron imaging (qBEI) was performed on paired iliac crest biopsies (risedronate, n = 18; placebo, n = 13) before and after treatment, and the mineral volume fraction in the trabecular bone was calculated. Combining dual-energy X-ray absorptiometric values with the mineral volume fraction for the same patients allowed us to calculate the relative change in trabecular bone volume with treatment. This showed that the effect on BMD was likely to be due partly to changes in matrix mineralization and partly due to changes in bone volume. After treatment, trabecular bone volume in the lumbar spine tended to increase in the risedronate group (+2.4%, nonsignificant) but there was a significant decrease (-3.7%, P < 0.05) in the placebo group. Calcium supplementation with adequate levels of vitamin D led to an approximately 3.3% increase in mineral content in the bone material independently of risedronate treatment. This increase was larger in patients with lower matrix mineralization at baseline and likely resulted from correction of calcium/vitamin D deficiency as well as from reduced bone remodeling. Combining BMD and bone mineralization density distribution data show that in postmenopausal osteoporosis 3-year treatment with risedronate preserves or may increase trabecular bone volume, unlike placebo. This analysis also allows, for the first time, separation of the contributions of bone volume and matrix mineralization to the increase in BMD.

Bone Remodeling Rate and Remodeling Balance Are Not Co-Regulated in Adulthood: Implications for the Use of Activation Frequency as an Index of Remodeling Rate

Use of activation frequency as a measure of remodeling rate assumes co-regulation of remodeling rate and remodeling balance. In iliac crest biopsy specimens from 57 healthy subjects 19-80 yr of age, no correlations were shown between these variables, an observation that challenges the use of activation frequency as an estimate of remodeling rate. The histomorphometric derivation of activation frequency assumes that the remodeling rate is dependent on the duration of the remodeling cycle and the amount of bone formed in individual remodeling units. This implies that remodeling balance and remodeling rate are co-regulated. We tested this assumption in normal human adult cancellous bone. Relationships between indices of bone formation at the basic multicellular unit (BMU) level (wall width and mineral apposition rate) and indices of remodeling rate (mineralizing perimeter and osteoid perimeter) were examined in iliac crest biopsies obtained from 57 healthy adults (24 men) 19-80 yr of age. Univariate analysis revealed a negative correlation between wall width and osteoid perimeter (r = -0.38; p = 0.0004), but there was no correlation between wall width and mineralizing perimeter or between mineral apposition rate and either mineralizing or osteoid perimeter. After adjustment for age and sex, the association between wall width and osteoid perimeter was no longer observed. Both wall width and mineral apposition rate correlated negatively with age (r = -0.75, p < 0.0001 and r = -0.27, p = 0.05, respectively). Our results indicate that remodeling balance and remodeling rate are not co-regulated in adult human bone. Activation frequency, as currently derived from histomorphometric variables, may therefore be unreliable as an indicator of remodeling rate.

Interrelationships Between Bone Microarchitecture and Strength in Ovariectomized Monkeys Treated With Teriparatide

Bone microarchitecture measured at the iliac crest at 6 mo was confirmed to be a reasonable surrogate for, and a predictor of, architecture and strength of the femoral neck and lumbar vertebra after 18 mo of teriparatide treatment. However, the data taken together showed the importance of cortical bone volume for vertebra to assess pharmacological effects on bone quality. Improvements in bone architecture with teriparatide treatment are suggested to contribute to fracture risk reduction in osteoporotic patients. Teriparatide significantly improves microarchitecture in the iliac crest of humans by stimulating bone modeling and remodeling processes that differ dramatically from those induced by antiresorptives. The relationship between improvements of bone microarchitecture and improvements of bone strength with teriparatide treatment has not yet been fully studied. Ovariectomized monkeys were administered vehicle (n = 20); teriparatide 1.0 microg/kg/d (n = 19); or teriparatide 5.0 microg/kg/d (n = 21) for 18 mo. Iliac crest biopsies were obtained at 6 and 15 mo after initiation of treatment. Animals were killed after 18 mo of treatment, and adjacent vertebrae or contralateral proximal femora were processed for biomechanical or histomorphometric analyses. Pearson correlation analyses were performed to assess the relationship between biomechanical and static histomorphometric parameters of lumbar vertebra, femoral neck, and iliac crest biopsies. Static histomorphometric parameters of the 6- and 15-mo biopsies were significantly correlated with the vertebral and femoral neck parameters obtained at 18 mo of teriparatide treatment. Iliac crest biopsy parameters at 6 and 15 mo also correlated with vertebral and femoral neck strength at 18 mo. Static histomorphometry of the lumbar vertebra and femoral neck at 18 mo also significantly correlated with strength at these sites. However, cortical bone volume of the lumbar vertebrae had the strongest correlation with vertebral and femoral neck strength (r = 0.74 and 0.71, respectively). Teriparatide dose dependently improved cortical and trabecular microarchitecture of vertebra and femoral neck, as well as trabecular microarchitecture of the iliac crest. Bone microarchitecture at all sites was significantly correlated with lumbar vertebra and femoral neck strength. Cortical bone volume of vertebra had the strongest correlation with vertebral and femoral neck strength. Therefore, structural improvement seemed to be part of the mechanism for improved strength observed with teriparatide treatment. Trabecular bone architecture of the iliac crest at 6 mo also correlated with vertebral and femoral neck strength, as did femoral neck (cortical and trabecular) histomorphometry and trabecular histomorphometry of vertebra after 18 mo of treatment. Because clinical assessment of cortical bone volume is not readily possible for vertebra noninvasively, these findings confirm the importance of iliac crest biopsies to monitor skeletal health and show that biopsies are a reasonable surrogate to assess spine and femoral neck structure and function.

Osteopenia, increased fracture risk and improvement in bone density with the use of Bisphosphonates in patients with Pompe disease

age. The causes for age related bone loss are multifactorial and the risk factors most likely have amuch earlier onset during bone development. The peak bone mass, reached between the ages of 25–30 is crucial for the future fracture risk. A small number of young patients develop early bone loss or an inability to fully build the skeleton. The goal of our investigation was to characterize juvenile osteoporosis and find histomorphological differences between juvenile osteoporosis and senile osteoporosis. Methods: Iliac crest biopsies, embedded into MMA, from patients younger than 30 years of age with clinical manifestation of osteopenia were analyzed. 344 male and 284 female patients were compared with data from a total of 21000 biopsies from osteoporotic patients of all ages. We evaluated medication, primary diseases, histological bone turnover and osteoid surfaces, as well as hematopoietic disorders. Results: 3% of all 21000 patients in the register were younger than 30 years of age. The portion of male patients was slightly higher (1.6% vs.1.4%). Histological bone turnover showed 40% of the patients had low turnover osteoporosis (LTO), 14% of the patients high turnover osteoporosis (HTO), and 46% had a normal and balanced turnover. 8.8% had an additional osteomalacic component. 6.7% had reactive bone marrow changes. 8.5% had corticosteroid induced bone loss. Rare primary causes for juvenile osteoporosis were as follows: M. Cushing, systemic mastocytosis, osteogenesis imperfecta, (pseudo-) hypoparathyroidism, dysplastic osteopathy, hypogonadism, primary hyperparathyroidism, gout-oxalate-osteopathy, Bamatter-syndrome and hyperthyrosis. 11 female patients received bisphosphonate therapy. Discussion: Patients with juvenile osteoporosis are rare and account for 3% of all cases we investigated. Less than 20% have an underlying disease or known risk factor that can be linked to osteopenia and referred to as secondary osteoporosis. Over 80% of children and young adults with bone loss have to be considered as idiopathic based on the histological diagnosis. Surprisingly, the number of LTO outweighs HTO conditions by 36% leading to the assumption that a low remodeling rate with little bone formation accounts for a negative balance in the bone mineral units during growth. Osteomalacic components are not more frequent than in the older population. Reactive bone marrow changes, e.g. due to anorexia nervosa, is 2–3% higher than in the total population, indicating a possible nutritive cause for osteopenia in more patients. Malignant hematological disorders were not present. Only 1.8% received a bone specific treatment.

Megakaryocyte Size and Concentration in the Bone Marrow of Thrombocytopenic and Nonthrombocytopenic Neonates

Thrombocytopenia is frequent among sick neonates, but little is known about its underlying mechanisms. It is known, however, that neonatal megakaryocytes are smaller and of lower ploidy than their adult counterparts and that smaller megakaryocytes produce fewer platelets than larger, more polyploid, megakaryocytes. We hypothesized that neonatal megakaryocytes would not increase their size in response to thrombocytopenia, thus limiting the ability of neonates to mount a response. To test this, we obtained marrow specimens from thrombocytopenic and nonthrombocytopenic neonates and adults. Megakaryocytes were immunohistochemically stained, quantified using an eyepiece reticle, and measured using an image analysis system with incorporated electronic micrometer. We found that, after adjusting for differences in cellularity, neonates and adults had similar megakaryocyte concentrations. When samples from the same sources were compared (tibial clot and vertebral body sections in neonates, iliac crest biopsies in adults), there were also no differences in megakaryocyte concentration between thrombocytopenic and nonthrombocytopenic subjects. The megakaryocyte diameter, however, was greater in adults than in neonates (19.4 +/- 3.0 versus 15.3 +/- 1.7 microm, p<0.0001). Thrombocytopenic adults also had a higher proportion of large megakaryocytes than nonthrombocytopenic adults (p<0.001). This was not observed among thrombocytopenic neonates, suggesting a developmental limitation in their ability to increase megakaryocyte size.

Effects of Continuing or Stopping Alendronate After 5 Years of Treatment. The Fracture Intervention Trial Long-term Extension (FLEX): A Randomized Trial

Pharmacokinetic studies have shown that bisphosphonates remain in the bone matrix for many years and are gradually released as bone is resorbed. The terminal half-life of one of these drugs, alendronate, exceeds 10 years, raising the possibility that some of its skeletal effects may continue for years after treatment ceases. In the Fracture Intervention Trial, a randomized, double-blind, placebo-controlled trial conducted at 10 clinical centers in the US, 1,099 postmenopausal women who had taken alendronate for an average of 5 years were randomly assigned to take alendronate in a dose of 5 or 10 mg daily or placebo for 5 years longer. All participants had low bone mineral density (BMD) at the femoral neck at the outset. Follow-up for clinical fractures averaged 4.2 years. Adding 5 years of alendronate therapy in a daily dose of 5 or 10 mg maintained total hip BMD compared with placebo. The mean total hip BMD declined 1% in women taking the medication and 3.4% in placebo recipients. Similar changes were found for the femoral neck and trochanter when analyzed separately. Lumbar spine BMD increased by 5.3% in the alendronate group and 1.5% in placebo patients. Comparable differences were found for total-body and forearm BMD. At each site, the gain in BMD after 10 years on alendronate was significantly greater than when treatment was limited to 5 years. Markers of bone turnover remained close to baseline during continued alendronate therapy but increased gradually in women taking placebo. Despite a lack of significant differences between treatment groups for all clinical fractures or nonvertebral fractures, the risk of clinical vertebral fracture was significantly lower for women who continued on alendronate (2.4% versus 5.3% in placebo recipients). The relative risk was 0.45, with a 95% confidence interval of 0.24-0.85. Loss of height did not differ significantly. No histomorphometric differences were found when 18 iliac crest biopsies were examined. Women in the two groups had similar numbers of serious adverse effects, and there were no significant group differences in rates of withdrawal because of adverse events or in mortality rates. Continuing alendronate therapy for as long as 10 years maintains bone mass, reduces bone remodeling, and does not increase the risk of fracture. Maintaining active treatment may be especially beneficial for women who are at high risk of clinical vertebral fracture as evidenced by a history of fracture or very low BMD.

Reduced bone formation and relatively increased bone resorption in absorptive hypercalciuria

Absorptive hypercalciuria (AH), a common stone-forming condition characterized biochemically by intestinal hyperabsorption of calcium and hypercalciuria may be associated with bone loss. In AH type I (AH-1), hypercalciuria persists despite restriction in dietary calcium intake. We therefore hypothesized that the skeleton may contribute to the hypercalciuria in this subgroup of patients. Histomorphometric analysis of iliac crest biopsies were performed on nine stone-formers with AH-1 and on nine matched normal subjects. After stabilization on a stone-prevention diet, calcium homeostasis in the stone formers was then evaluated on inpatient constant metabolic diet before and after short-term blockade of bone resorption by alendronate (10 mg daily, 17 days total). Compared with controls, the stone-formers had lower indices of bone formation (osteoblast surface/bone surface 1.8+/-2.1 vs 3.0+/-1.5%, P=0.04; wall thickness 35.8+/-6.9 vs 47.2+/-7.6%, P=0.001) and relatively higher bone resorption (osteoclast surface/bone surface 0.4+/-0.2 vs 0.2+/-0.2%, P=0.05). In the stone-formers, a short-term course of alendronate treatment corrected fasting urinary calcium (0.14+/-0.06 to 0.06+/-0.04 mg Ca/mg Cr, P=0.001) and marginally reduced 24-h urinary calcium by 48 mg/day (P=0.06). Increased intestinal calcium absorption and hypercalciuria persisted, but estimated calcium balance improved (P=0.007). Our results suggest that the hypercalciuria of AH-1 originates primarily from intestinal hyperabsorption of calcium, but bone resorption in excess of bone formation may contribute.

Use of isotopes in the diagnosis of hematopoietic disorders

Isotopes of iron have been paramount in clinical tracer studies of hematopoiesis. Kinetics of disappearance from plasma, appearance in circulating erythrocytes, and localization in general areas of bone marrow, spleen, and liver were early revealed by gamma-/beta-emitting Fe-59 with note of characteristic differences of kind and degree of dysfunction among various anemias. Findings have helped in management (e.g., decisions about splenectomy). Positron/gamma-emitting Fe-52 (better for imaging) has provided more detail on marrow expansion and erythropoietic relocation. Other gamma-emitting tracers of hematopoiesis have been In-111 (linked to transferrin) and technetium 99m (Tc-99m) colloids (localizing to reticuloendothelial cells, which have close association with blood cell progenitors), but Fe-52 has proved to be more accurate for recognition of erythropoiesis. Occasional diverse heterotopic sites of hematopoiesis beyond spleen and liver in states of bone marrow disease have posed diagnostic challenges and also raised questions about migration and/or activation of stem cells. Studies of granulopoiesis utilize Tc-99m--labeled leukocytes or Tc99m-labeled antibodies to circulating and progenitor white cells. Iron-deficiency anemias due to malnutrition, malabsorption, blood loss, or special need are explored by dual study (oral vs intravenous) with radioactive (Fe-59, Fe-55) or stable (Fe-54, Fe-57) iron isotopes, which can guide dietary supplementation. Tests for B-12 deficiency in pernicious anemia or malabsorption with radioisotopes of cobalt (Co-57, Co-58) have been upgraded in sensitivity and scope. Rates of oxidation to expired carbon dioxide from particular carbon (radioactive C-14 or stable C-13)--labeled compounds can test B-12 or folic acid deficiencies or gastric infestation as causes of megaloblastic anemias.

Treatment of Skeletally Mature Ovariectomized Rhesus Monkeys With PTH(1-84) for 16 Months Increases Bone Formation and Density and Improves Trabecular Architecture and Biomechanical Properties at the Lumbar Spine

Histomorphometric studies of treatments for osteoporosis in humans are restricted to iliac crest biopsies. We studied the effects of PTH(1-84) treatment at the lumbar spine of skeletally mature ovariectomized rhesus monkeys. PTH increased bone turnover, rapidly normalized BMD, and increased vertebral compressive strength. PTH increased trabecular bone volume primarily by increasing trabecular number by markedly increasing intratrabecular tunneling. Histomorphometric studies of the anabolic properties of PTH(1-84) (PTH) and related peptides in human bone are restricted to iliac crest biopsies. The ovariectomized (OVX) monkey is an accepted model of human postmenopausal bone loss and was used to study the effects of PTH treatment at clinically relevant skeletal sites. Skeletally mature rhesus monkeys were OVX or sham-operated and, after a bone depletion period of 9 months, treated daily for 16 months with PTH (5, 10, or 25 microg/kg). Markers of bone formation (serum osteocalcin) and resorption (urine N-telopeptide [NTX]) and lumbar spine BMD were measured throughout the study. Trabecular architecture and vertebral biomechanical properties were quantified at 16 months. PTH treatment induced dose-dependent increases in bone turnover but did not increase serum calcium. Osteocalcin was significantly increased above OVX controls by 1 month. NTX was significantly elevated at 1 month with the highest dose, but not until 12 months with the 5 and 10 microg/kg doses. Lumbar spine BMD was 5% lower in OVX than in sham animals when treatment was started. All PTH doses increased BMD rapidly, with sham levels restored by 3-7 months with 10 and 25 microg/kg and by 16 months with 5 microg/kg. PTH treatment increased trabecular bone volume (BV/TV), primarily by increasing trabecular number, and dose-dependently increased bone formation rate (BFR) solely by increasing mineralizing surface. The largest effects on BV/TV and yield load occurred with the 10 microg/kg dose. The highest dose reduced trabecular thickness by markedly increasing intratrabecular tunneling. PTH treatment of OVX rhesus monkeys increased bone turnover and increased BV/TV, BMD, and strength at the lumbar spine. All PTH doses were safe, but the 10 microg/kg dose was generally optimal, possibly because the highest dose resulted in too marked a stimulation of bone remodeling.

Bone Material Properties in Trabecular Bone From Human Iliac Crest Biopsies After 3- and 5-Year Treatment With Risedronate

Long-term effects of risedronate on bone mineral maturity/crystallinity and collagen cross-link ratio in triple iliac crest biopsies of osteoporotic women were evaluated. In this double-blinded study, 3- and 5-year treatment with risedronate arrested the tissue aging encountered in untreated osteoporosis and in osteoporosis treated with other antiresorptives. This effect may be contributing to risedronate's antifracture efficacy. Risedronate is widely used in the treatment of osteoporosis. It reduces bone turnover, increases BMD, and decreases fracture risk. To date, there are no data available on the long-term effects of risedronate on bone material properties in humans. Osteoporotic women enrolled in the VERT-NA trial received either risedronate (5 mg/day, orally) or placebo for up to 5 years. All subjects received calcium. They also received vitamin D supplementation if deficient at baseline. Triple iliac crest biopsies were collected from a subset of these subjects at baseline, 3 years, and 5 years. Mineral maturity/crystallinity and collagen cross-link ratio was measured in these biopsies using Fourier transform infrared imaging. Patients that received placebo exhibited increased mineral maturity/crystallinity and collagen cross-link ratio after 3 and 5 years compared with baseline values. On the contrary, patients that received risedronate retained baseline values in both bone material indices throughout. A more spatially detailed analysis revealed that this was achieved mainly through beneficial effects on active bone-forming areas. Surprisingly, patients that received risedronate achieved premenopausal values at bone-forming areas in both indices after 5 years of treatment. Long-term treatment with risedronate affects bone material properties (mineral maturity/crystallinity and collagen cross-link ratio) and arrests the tissue aging apparent in untreated osteoporosis. These changes at the material level of the bone matrix may contribute to risedronate's rapid and sustained antifracture efficacy in osteoporotic patients.

Bone tissue compositional differences in women with and without osteoporotic fracture

It is generally accepted that the hallmark of osteoporosis is a reduction in bone mass. There is significant overlap, however, in bone mineral density between osteoporotic and normal individuals. This study examined the chemical composition of bone tissue obtained from women who had sustained a fracture and women without fracture to determine if there are differences between the two groups. Nineteen fractured and eleven non-fractured proximal femurs were obtained, matched for age and bone volume fraction obtained from micro-computed tomography. Trabecular bone specimens were examined by Raman spectroscopy to determine measures of chemical composition. A subset of the specimens was utilized to compare locations at the fracture and regions at least 2 mm away from apparent tissue damage using Raman spectroscopy. In addition, fifteen iliac crest biopsies each were obtained from women who had sustained a fracture and from normal controls. Raman spectroscopy was used to determine measures of chemical composition of trabecular and cortical bone. The results demonstrated that femoral bone tissue in the region of visible damage had a trend towards differences compared to regions at least 2 mm from visible damage. Femoral trabecular bone in fractured women had a higher carbonate/amide I area ratio than in unfractured women. Iliac crest biopsies revealed a higher carbonate/phosphate ratio in cortical bone from women who had sustained a fracture. Results suggest that the chemical composition of bone tissue may be an additional risk factor for osteoporotic fracture.

Effects of 3- and 5-Year Treatment With Risedronate on Bone Mineralization Density Distribution in Triple Biopsies of the Iliac Crest in Postmenopausal Women

Long-term effects of risedronate on bone mineralization density distribution in triple transiliac crest biopsies of osteoporotic women were evaluated. In this double-blinded study, 3- and 5-year treatment with risedronate increased the degree and homogeneity of mineralization without producing hypermineralization. These changes at the material level of bone could contribute to risedronate's antifracture efficacy. Risedronate, a nitrogen-containing bisphosphonate, is widely used in the treatment of osteoporosis. It reduces bone turnover, increases BMD, and decreases fracture risk. To date, there are no data available on the long-term effects of risedronate on bone mineralization density distribution (BMDD) in humans. Osteoporotic women enrolled in the VERT-NA trial received either risedronate (5 mg/day, orally) or placebo for up to 5 years. All subjects received calcium and vitamin D supplementation if deficient at baseline. Triple iliac crest biopsies were collected from a subset of these subjects at baseline and 3 and 5 years. BMDD was measured in these biopsies using quantitative backscattered electron imaging, and the data were also compared with a normal reference group. At baseline, both risedronate and placebo groups had a lower degree and a greater heterogeneity of mineralization as well as an increase in low mineralized bone compared with the normal reference group. The degree of mineralization increased significantly in the risedronate as well as in the placebo group after 3- and 5-year treatment compared with baseline. However, the degree of mineralization did not exceed that of normal. Three-year treatment with risedronate significantly increased the homogeneity of mineralization and slightly decreased low mineralized bone compared with placebo. Surprisingly with 5-year risedronate treatment, heterogeneity of mineralization increased compared with 3-year treatment, which might indicate an increase in newly formed bone. Long-term treatment with risedronate affects the homogeneity and degree of mineralization without inducing hypermineralization of the bone matrix. These changes at the material level of the bone matrix may contribute to risedronate's antifracture efficacy in osteoporotic patients.

A Novel Tetracycline Labeling Schedule for Longitudinal Evaluation of the Short-Term Effects of Anabolic Therapy With a Single Iliac Crest Bone Biopsy: Early Actions of Teriparatide

We describe a quadruple tetracycline labeling method that allows longitudinal assessment of short-term changes in bone formation in a single biopsy. We show that 1 month of hPTH(1-34) treatment extends the bone-forming surface, increases mineral apposition rate, and initiates modeling-based formation. Iliac crest biopsy, with histomorphometric evaluation, provides important information about cellular activity in bone. However, to obtain longitudinal information, repeat biopsies must be performed. In this study, we show the capability to obtain short-term longitudinal information on bone formation in a single biopsy using a novel, quadruple labeling technique. Two tetracycline labels were administered using a standard 3 days on, 12 days off, 3 days on format. Four weeks later, the tetracycline labeling was repeated using the same schedule but with a different tetracycline that can be distinguished from the first by its color under fluorescent light. Iliac crest biopsies were performed 1 week later and prepared undecalcified for histomorphometry. Indices of bone formation 1 month apart were measured and calculated using the two sets of labels. We used this method to investigate the early effects of teriparatide [hPTH(1-34)] treatment on bone formation. The results were compared with those from a group of control subjects who were quadruple-labeled, but did not receive hPTH(1-34). Treatment with hPTH(1-34) dramatically stimulated bone formation on cancellous and endocortical surfaces. This was achieved by both an increase in the linear rate of matrix apposition and extension of the bone-forming surface. New bone was deposited on previously quiescent surfaces (i.e., modeling-based formation), but a proportion of this could occur by encroachment from adjacent resorption cavities. A single transiliac crest bone biopsy, after sequential administration of two sets of tetracycline labels is a useful approach to study the short-term effects of anabolic agents on human bone. One month of hPTH(1-34) treatment extends the bone-forming surface, increases mineral apposition rate, and initiates modeling-based formation.

Ageing and human bone collagen synthesis

A loss of bone mineral density and collagen concentration occurs with ageing, but possible deficits in human bone collagen synthesis have not previously been examined. We studied 4 healthy elderly men (age 62 ± 7 y) and 2 healthy postmenopausal elderly women (55,62y) in the post absorptive state and compared the results with those from our previously published data for young healthy men. Subjects fasted overnight, were given a flooding dose of 1-13C proline over 3 min with an iliac crest biopsy being taken under local anesthesia and mild sedation 120 min later. There were no differences in the rate of immature bone collagen synthesis between the elderly and young men (NaCl fraction: 0.57±0.18 v 0.57±0.10 %.h−1; HOAc fraction: 0.23±0.10 v 0.24±0.05 %.h−1 respectively), but the rate of mature bone collagen synthesis was less in the elderly than the young men (pepsin fraction: 0.04±0.01 v 0.07±0.01 %.h−1, P< 0.002; hot water fraction: 0.04±0.01 v 0.06±0.01 %.h−1, P<0.002, respectively). In elderly women the pattern of rates of collagen synthesis was different to that in men with lower rates of immature collagen synthesis than in the young men (NaCl: 0.36 %.h−1; HOAc: 0.17 %.h−1) and higher rates of mature collagen synthesis (pepsin: 0.08 %.h−1; hot water: 0.08 %.h−1). Ageing in men does not appear to effect osteoblasts ability to synthesis collagen but extracellular processing into fibrillar collagen appears to be defective, whereas in women procollagen synthesis may be decreased. Supported by The Wellcome Trust, Diabetes UK, UK MRC, BBSRC.