Bone histomorphometry in transiliac biopsies from 48 normal, healthy men.

Investigators and clinicians use bone histomorphometry data from iliac bone biopsies to study bone abnormalities in diseased patients, and to understand the safety and effectiveness of pharmaceutical interventions. This requires access to a high quality normal data-set to be used for comparisons, a resource that has not been adequate to date. The objective of this work is to present static and dynamic bone histomorphometry data from transilial bone biopsies performed on 48 healthy males, evenly distributed between ages 45 and 75. In addition, we compared these results with results from our earlier study in normal postmenopausal women (Recker et al., 1988 [1]). The data include bone density and anthropometric measurements, micro-CT, and a collection of serum biochemical measurements. We found that several of the histomorphometry variables were correlated with serum measurements, i.e. serum testosterone and sex hormone-binding globulin (SHBG). Micro-CT variables were correlated with the static histomorphometry variables, and were very similar. Age-related changes were observed for both histomorphometry and Micro-CT, but were surprisingly small in most cases. Comparisons with our previously reported histomorphometry data from normal women were surprisingly similar, but there was a significant age by gender interaction in the wall thickness (W.Th) measurements, i.e. there was a small increase in this variable with age in men, and a significant decline with age in women. The population selected for this study, and the prior study in normal women, were carefully chosen so as to rule out the presence of clinical, life-style or other confounding factors. While the cohort chosen herein was a convenience sample, and not a population-based sample, we believe it can be used as a reference standard with proper precautions in its interpretation and in its comparisons with diseased populations.

Case report: Electron microscopic evaluation of bone from a patient treated with cinacalcet hydrochloride, maxacalcitol, and alfacalcidol for hyperparathyroid bone disease with secondary hyperparathyroidism.

Evaluation of bone is of great importance in chronic kidney disease patients, as these patients are at an increased risk for fractures. We treated a hemodialysis patient suffering from hyperparathyroid bone disease with cinacalcet hydrochloride and concurrent administration of maxacalcitol and alfacalcidol for a year. Hyperparathyroid bone disease is characterized by cortical thinning, increased cortical porosity, reduced trabecular bone volume, and increased hypomineralized matrix volume, and there is little information to date about the effects of treatment with cinacalcet hydrochloride on the bone fragility in patients with hyperparathyroid bone disease. In the present study, histological and backscattered electron microscopic evaluation of this combination treatment revealed an excellent improvement of both bone volume and bone morphology. This treatment improved cortical thinning, cortical porosity, and trabecular thinning. Furthermore, the treatment also reduced hypomineralized matrix volume, indicative of improved mineralization by osteocytes. We speculate that the intermittent maxacalcitol administration may have effectively stimulated the vitamin D receptors expressed on osteocytes and osteoblasts, resulting in increased mineralization. Our approach for evaluating the bone in patients with chronic kidney disease by backscattered electron microscopy is novel.

Bone matrix mineralization is preserved during early perimenopausal stage in healthy women: a paired biopsy study.

UNLABELLED: Bone matrix mineralization based on quantitative backscatter electron imaging remained unchanged during the first year of menopause in paired transiliac biopsy samples from healthy women. This suggests that the reported early perimenopausal reductions in bone mineral density are caused by factors other than decreases in the degree of mineralization. INTRODUCTION: It is unknown whether perimenopausal loss of bone mass is associated with a drop in bone matrix mineralization. METHODS: For this purpose, we measured the bone mineralization density distribution (BMDD) by quantitative backscatter electron imaging (qBEI) in n = 17 paired transiliac bone biopsy samples at premenopausal baseline and 12 months after last menses (obtained at average ages of 49 ± 2 and 55 ± 2 years, respectively) in healthy women. For interpretation of BMDD outcomes, previously measured bone mineral density (BMD) and biochemical and histomorphometric markers of bone turnover were revisited for the present biopsy cohort. RESULTS: Menopause significantly decreased BMD at the lumbar spine (-4.5 %) and femoral neck (-3.8 %), increased the fasting urinary hydroxyproline/creatinine ratio (+60 %, all p < 0.01) and histomorphometric bone formation rate (+25 %, p < 0.05), but affected neither cancellous nor cortical BMDD variables (paired comparison p > 0.05). Mean calcium concentrations of cancellous (Cn.CaMean) and cortical bone (Ct.CaMean) were within normal range (p > 0.05 compared to established reference data). Ct.CaMean was significantly correlated with Cn.CaMean before (R = 0.81, p < 0.001) and after menopause (R = 0.80, p < 0.001) and to cortical porosity of mineralized tissue (Ct.Po.) after menopause (R = -0.57, p = 0.02). CONCLUSIONS: Surprisingly, the BMDD was found not affected by the changes in bone turnover rates in this cohort. This suggests that the substantial increase in bone formation rates took place shortly before the second biopsy, and the bone mineralization changes lag behind. We conclude that during the first year after the last menses, the degree of bone matrix mineralization is preserved and does not contribute to the observed reductions in BMD.

Bone impairment in oxalosis: An ultrastructural bone analysis.

Deposition of calcium oxalate crystals in the kidney and bone is a hallmark of systemic oxalosis. Since the bone compartment can store massive amounts of oxalate, patients present with recurrent low-trauma fractures, bone deformations, severe bone pains and specific oxalate osteopathy on plain X-ray. Bone biopsy from the iliac crest displays specific features such as oxalate crystals surrounded by a granulomatous reaction due to an invasion of bone surface by macrophages. We present data obtained in 10 samples from 8 patients with oxalosis (16-68 years) who underwent iliac crest bone biopsy and bone quality analysis using modern methods (microradiography, microindentation, Fourier Transform InfraRed Microspectroscopy, transmission electron microscopy) in addition to histomorphometry. Disseminated calcium oxalate deposits (whewellite) were found in the bone marrow space (with a granulomatous reaction) but not in the bone matrix. Calcium oxalate deposits were totally surrounded by macrophages and multinucleated giant cells, and a phagocytosis activity was sometimes observed. Very few calcium oxalate crystals were directly in close contact with the mineral substance of the bone. Bone mineralization was not modified by the presence of calcium oxalate even in close vicinity. Bone quality analysis also revealed a harder bone than normal, perhaps in relationship with decreased carbonate content in the mineral. This increase in bone hardness could explain a more "brittle" bone. In patients with oxalosis, the formation and growth of calcium oxalate crystals in the bone appeared independent of apatite. The mechanisms leading to nucleation and growth of oxalate deposits are still unclear and deserve further studies.

Age-related changes in vertebral and iliac crest 3D bone microstructure--differences and similarities.

UNLABELLED: Age-related changes of vertebra and iliac crest 3D microstructure were investigated, and we showed that they were in general similar. The 95th percentile of vertebral trabecular thickness distribution increased with age for women. Surprisingly, vertebral and iliac crest bone microstructure was only weakly correlated (r = 0.38 to 0.75), despite the overall similar age-related changes. INTRODUCTION: The purposes of the study were to determine the age-related changes in iliac and vertebral bone microstructure for women and men over a large age range and to investigate the relationship between the bone microstructure at these skeletal sites. METHODS: Matched sets of transiliac crest bone biopsies and lumbar vertebral body (L2) specimens from 41 women (19-96 years) and 39 men (23-95 years) were micro-computed tomography (µCT) scanned, and the 3D microstructure was quantified. RESULTS: For both women and men, bone volume per total volume (BV/TV), connectivity density (CD), and trabecular number (Tb.N) decreased significantly, while structure model index (SMI) and trabecular separation (Tb.Sp) increased significantly with age at either skeletal site. Vertebral trabecular thickness (Tb.Th) was independent of age for both women and men, while iliac Tb.Th decreased significantly with age for men, but not for women. In general, the vertebral and iliac age-related changes were similar. The 95th percentile of the Tb.Th distribution increased significantly with age for women but was independent of age for men at the vertebral body, while it was independent of age for either sex at the iliac crest. The Tb.Th probability density functions at the two skeletal sites became significantly more similar with age for women, but not for men. The microstructural parameters at the iliac crest and the vertebral bodies were only moderately correlated from r = 0.38 for SMI in women to r = 0.75 for Tb.Sp in men. CONCLUSION: Age-related changes in vertebral and iliac bone microstructure were in general similar. The iliac and vertebral Tb.Th distributions became more similar with age for women. Despite the overall similar age-related changes in trabecular bone microstructure, the vertebral and iliac bone microstructural measures were only weakly correlated (r = 0.38 to 0.75).

Multipotential stromal cell abundance in cellular bone allograft: comparison with fresh age-matched iliac crest bone and bone marrow aspirate.

AIM: To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allograft and compare with fresh age-matched iliac crest bone and bone marrow (BM) aspirate. MATERIALS & METHODS: MSC characterization used functional assays, confocal/scanning electron microscopy and whole-genome microarrays. Resident MSCs were enumerated by flow cytometry following enzymatic extraction. RESULTS: Allograft material contained live osteocytes and proliferative bone-lining cells defined as MSCs by phenotypic and functional capacities. Without cultivation/expansion, the allograft displayed an 'osteoinductive' molecular signature and the presence of CD45(-)CD271(+)CD73(+)CD90(+)CD105(+) MSCs; with a purity over 100-fold that of iliac crest bone. In comparison with BM, MSC numbers enzymatically released from 1 g of cellular allograft were equivalent to approximately 45 ml of BM aspirate. CONCLUSION: Cellular allograft bone represents a unique nonimmune material rich in MSCs and osteocytes. This osteoinductive graft represents an attractive alternative to autograft bone or composite/synthetic grafts in orthopedics and broader regenerative medicine settings.

Early effects of zoledronic acid and teriparatide on bone microarchitecture, remodeling and collagen crosslinks: comparison between iliac crest and lumbar vertebra in ewes.

Iliac crest bone biopsies are used to assess the mechanism of action of drug treatments, yet there are little data comparing this site to sites prone to fracture. The purpose of this study was to compare the delay and the amplitude of responses to treatment in two different bone sites. The short-term effects of zoledronic acid and teriparatide on microarchitecture, collagen crosslinks and bone remodeling were evaluated in iliac crest and lumbar vertebrae. Aged ewes (n=8/gr) received either vehicle (CTRL) or a single injection of zoledronic acid (ZOL, 10mg) or daily injections of teriparatide (TPTD, 20 µg/d) for 3 months. Blood samples were collected monthly for assessing bone turnover markers. At the end of the study, a transiliac bone biopsy (IC) and L1 lumbar vertebrae (LV1) were collected to assess bone microarchitecture; pyridinoline (PYD), deoxypyridinoline (DPD), pentosidine (PEN) content, static and dynamic parameters of bone remodeling. In CTRL, Tb-BV/TV was significantly higher in LV1 than IC (p<0.0001). This was associated with a trend of higher Tb.N, Tb.Th, DA, an inferior Conn.D and a lower bone turnover as shown by the decreases of osteoid parameters, MS/BS, Ac.f in LV1 when compared to IC. In addition, the ratio PYD/DPD was 4 times higher in LV1 than IC. After 3 months, significant decreases of sALP (p<0.001) and sCTX (p<0.001) were observed in the ZOL-group whereas in TPTD-group, after transient increases, they returned to baseline values. When compared to their respective CTRL, ZOL induced significant increases in Tb.BV/TV, Conn.D, Tb.N and Tb.Sp, in IC but not in LV1. Regardless of the site, ZOL markedly depressed the bone turnover: The static parameters of bone formation significantly decreased and the diminution of MS/BS, BFR/BS and Ac.f varied from -94 to -98% vs CTRL (p<0.01 to 0.001). It was associated with a diminution of the DPD content and the PYD/DPD ratio mainly in IC cortices. In contrast, after 3 months, TPTD did not modify the 3D structure and microarchitecture in IC and LV1, except a trend of higher Conn.D in IC, compared to IC-CTRL. TPTD treatment induced a significant increase in cortical porosity in LV1 (p<0.05) when compared to LV1-CTRL. Static parameters of bone formation and resorption were augmented in both sites, significantly only in LV1 (p<0.05) with a trend of increases in MS/BS and BFR/BS, compared to LV1-CTRL. In conclusion, in adult ewes, the bone mass, microarchitecture, remodeling and collagen crosslink content differ according to the bone site (iliac crest and vertebra). Furthermore, after 3 months, the responses to ZOL and TPTD were of different magnitude and delay between the two bone sites. The distinction of bone sites to study the early effects of anti-osteoporotic therapies appears meaningful in order to approach their site-specific anti-fracture efficacy.

Trabecular bone histomorphometry in humans with Type 1 Diabetes Mellitus.

Patients with Type 1 Diabetes Mellitus (DM) have markedly increased risk of fracture, but little is known about abnormalities in bone microarchitecture or remodeling properties that might give insight into the pathogenesis of skeletal fragility in these patients. We report here a case-control study comparing bone histomorphometric and micro-CT results from iliac biopsies in 18 otherwise healthy subjects with Type 1 Diabetes Mellitus with those from healthy age- and sex-matched non-diabetic control subjects. Five of the diabetics had histories of low-trauma fracture. Transilial bone biopsies were obtained after tetracycline labeling. The biopsy specimens were fixed, embedded, and scanned using a desktop µCT at 16 µm resolution. They were then sectioned and quantitative histomorphometry was performed as previously described by Recker et al. [1]. Two sections, >250 µm apart, were read from the central part of each biopsy. Overall there were no significant differences between diabetics and controls in histomorphometric or micro-CT measurements. However, fracturing diabetics had structural and dynamic trends different from nonfracturing diabetics by both methods of analysis. In conclusion, Type 1 Diabetes Mellitus does not result in abnormalities in bone histomorphometric or micro-CT variables in the absence of manifest complications from the diabetes. However, diabetics suffering fractures may have defects in their skeletal microarchitecture that may underlie the presence of excess skeletal fragility.

Abnormal bone microarchitecture and evidence of osteoblast dysfunction in premenopausal women with idiopathic osteoporosis.

CONTEXT: Idiopathic osteoporosis (IOP) in premenopausal women is an uncommon disorder of uncertain pathogenesis in which fragility fractures occur in otherwise healthy women with intact gonadal function. It is unclear whether women with idiopathic low bone mineral density and no history of fragility fractures have osteoporosis. OBJECTIVE: The objective of the study was to elucidate the microarchitectural and remodeling features of premenopausal women with IOP. DESIGN: We performed transiliac biopsies after tetracycline labeling in 104 women: 45 with fragility fractures (IOP), 19 with idiopathic low bone mineral density (Z score ≤-2.0) and 40 controls. Biopsies were analyzed by two-dimensional quantitative histomorphometry and three-dimensional microcomputed tomography. Bone stiffness was estimated using finite element analysis. RESULTS: Compared with controls, affected women had thinner cortices; fewer, thinner, more widely separated, and heterogeneously distributed trabeculae; reduced stiffness; and lower osteoid width and mean wall width. All parameters were indistinguishable between women with IOP and idiopathic low bone mineral density. Although there were no group differences in dynamic histomorphometric remodeling parameters, serum calciotropic hormones, bone turnover markers, or IGF-I, subjects in the lowest tertile of bone formation rate had significantly lower osteoid and wall width, more severely disrupted microarchitecture, lower stiffness, and higher serum IGF-I than those in the upper two tertiles, suggesting that women with low turnover IOP have osteoblast dysfunction with resistance to IGF-I. Subjects with high bone turnover had significantly higher serum 1,25 dihydroxyvitamin D levels and a nonsignificant trend toward higher serum PTH and urinary calcium excretion. CONCLUSIONS: These results suggest that the diagnosis of IOP should not require a history of fracture. Women with IOP may have high, normal or low bone turnover; those with low bone turnover have the most marked deficits in microarchitecture and stiffness. These results also suggest that the pathogenesis of idiopathic osteoporosis is heterogeneous and may differ according to remodeling activity.

Respective roles of organic and mineral components of human cortical bone matrix in micromechanical behavior: an instrumented indentation study.

Bone is a multiscale composite material made of both a type I collagen matrix and a poorly crystalline apatite mineral phase. Due to remodeling activity, cortical bone is made of Bone Structural Units (BSUs) called osteons. Since osteon represents a fundamental level of structural hierarchy, it is important to investigate the relationship between mechanical behavior and tissue composition at this scale for a better understanding of the mechanisms of bone fragility. The aim of this study is to analyze the links between ultrastructural properties and the mechanical behavior of bone tissue at the scale of osteon. Iliac bone biopsies were taken from untreated postmenopausal osteoporotic women, embedded, sectioned and microradiographed to assess the degree of mineralization of bone (DMB). On each section, BSUs of known DMB were indented with relatively high load (~500 mN) to determine local elastic modulus (E), contact hardness (H(c)) and true hardness (H) of several bone lamellae. Crystallinity and collagen maturity were measured by Fourier Transform InfraRed Microspectroscopy (FTIRM) on the same BSUs. Inter-relationships between mechanical properties and ultrastructural components were analyzed using multiple regression analysis. This study showed that elastic deformation was only explained by DMB whereas plastic deformation was more correlated with collagen maturity. Contact hardness, reflecting both elastic and plastic behaviors, was correlated with both DMB and collagen maturity. No relationship was found between crystallinity and mechanical properties at the osteon level.

Normative data on mineralization density distribution in iliac bone biopsies of children, adolescents and young adults.

Bone mineralization density distribution (BMDD) as assessed by quantitative backscattered electron imaging (qBEI) in iliac crest bone biopsies has become in the last years a powerful diagnostic tool to evaluate the effect of metabolic bone diseases and/or therapeutic interventions on the mineralization status of the bone material. However until now, normative reference data are only available for adults. The aim of the present study is to close this gap and establish normative data from children and compare them with reference BMDD data of adults. qBEI analyses were performed on bone samples from 54 individuals between 1.5 and 23 years without metabolic bone diseases, which were previously used as study population to establish normative histomorphometric standards. In the trabecular compartment, none of the BMDD parameters showed a significant correlation with age. The BMDD was shifted towards lower mineralization density (CaMean -5.6%, p<0.0001; CaPeak -5.6%, p<0.0001; CaLow +39.0% p<0.001; CaHigh -80.7%, p<0.001) and the inter-individual variation was higher compared to the adult population. The cortices appeared to be markedly less mineralized (CaMean -3.1%, p<0.0001) than cancellous bone due to higher amounts of low mineralized secondary bone. However, the cortical BMDD parameters showed a strong correlation (r=0.38 to 0.85, with p<0.001 to<0.0001) with cancellous BMDD parameters. In conclusion, this study provides evidence that BMDD parameters in growing healthy subjects are relatively constant and that these data can be used as normative references in pediatrics osteology. The larger inter-individual variability compared to adults is most likely related to alterations of the bone turnover rate during growth.

Comparison of quantitative cancellous bone connectivity analyses at two- and three-dimensional levels in dialysis patients.

Assessment of cancellous bone connectivity has the potential to aid in predicting fracture risk. Today, cancellous bone connectivity is generally assessed using bone sections obtained from biopsy. However, how reliably such two-dimensional (2-D) analyses visualize the 3-D properties has not been evaluated. Biopsied iliac bone samples were obtained from 47 chronic hemodialysis patients. Bone samples were observed using a microfocus X-ray computed tomography (microCT) system en bloc, and the cancellous bone microstructure was quantitatively assessed at both the 2- and 3-D levels. Cancellous bone microarchitecture was successfully reconstructed from the data obtained by the microCT system. Most of the results from node-strut analysis (NSA) revealed no statistically significant correlations between the 2- and 3-D analyses, with the exception that the number of nodes (N.Nd/TV) showed a mild but significant correlation. In contrast, the marrow space star volumes (V*m) of the 2- and 3-D analyses were highly correlated. NSA parameters including N.Nd/TV showed significant correlations with V*m at the 3-D level. In conclusion, V*m values were similar in the 2- and 3-D analyses, while most of the 2-D NSA parameters did not reflect the 3-D ones. Since V*m and most of the NSA parameters were correlated in the 3-D analyses, 2-D NSA would seem to have serious limitations for the assessment of cancellous bone microstructural properties. Further studies will thus be needed to establish appropriate methods for assessing cancellous bone connectivity in clinical practice.

A histological and ultrastructural study of the iliac crest apophysis in Legg-Calve-Perthes disease.

BACKGROUND: Legg-Calve-Perthes disease (LCPD) is a common hip disorder in children characterized by avascular necrosis of the proximal capital femoral epiphysis. The underlying etiology of the vascular disturbance is still unknown, but it is suggested that LCPD may be a part of a generalized constitutional disorder associated with growth disturbance of bone and cartilage tissue. In this study, the biopsy specimens of the iliac crest apophysis from LCPD patients were examined histologically and ultrastructurally to determine preexisting generalized abnormalities of endochondral ossification. METHODS: Iliac crest apophysis cartilage was taken during Salter innominate osteotomy from 11 children (8 boys and 3 girls) with LCPD at an average age of 7.8 years. As controls, the samples were also obtained from 10 children (2 boys and 8 girls) at an average age of 6.3 years undergoing Salter osteotomy due to residual acetabular dysplasia after reduction of developmental dysplasia of the hip. Each iliac crest apophysis specimen was examined histologically (Toluidine blue staining and Sudan III staining) and ultrastructurally. RESULTS: Although there were no obvious differences in Toluidine blue-stained sections of the iliac crest cartilage between LCPD and control patients, the Sudan III-positive chondrocytes in the resting cartilage were more prominent in the LCPD specimens than in the control specimens. These sudanophilic granules were confirmed to be lipid droplets by electron microscopic examinations. Ultrastructural examinations of the resting chondrocytes from 3 LCPD patients demonstrated numerous cytoplasmic inclusion bodies with electron dense materials, which were similar to those seen in some of the mucopolysaccharidoses. CONCLUSIONS: Increased lipid droplets and numerous cytoplasmic inclusions filled with fibrillar materials were suggestive of the initial metabolic changes of the chondrocytes, which may have a pivotal role in degenerating matrix and lead to vulnerability of the cartilage tissue. Our results indicated that generalized insufficiency in growth cartilage metabolism may be related to the onset of the disease in some LCPD patients.

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 (microCT) provide information on the three-dimensional (3D) microarchitecture of bone. We used microCT 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 microCT scanner (voxel size, 10.77 microm). Parameters measured in trabecular bone consisted of bone volume/tissue volume (BV/TV, %), direct trabecular thickness and trabecular spacing (Tb.Th and Tb.Sp, microm) 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 microCT 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 (mAbsDelta(ind)) 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, mAbsDelta(ind) 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 mAbsDelta(ind) 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 microCT and image analysis must comply with stringent quality criteria, especially the distance from the cortices must be standardized.

Cortical vascular canals in human mandible and other bones.

The human mandible is highly mineralized. We hypothesized that this is related to the local vascularity of the bone. This could not be examined directly, but, as a surrogate, intracortical vascular canal spaces of the human mandible were studied so that we could determine possible relationships with age, gender, location, dental status, and tissue mineralization. Canal numbers, area, and volume fraction were calculated from quantitative backscattered electron images of human mandibles aged 16-96 years. Data were compared with calvaria, maxilla, lumbar vertebra, femoral neck, and iliac crest. In the mandible, the buccal aspect of the midline was the most porous, the canals being larger and more numerous. The cortical porosity in the posterior of partially dentate mandibles was significantly greater than that of either dentate or edentate mandibles, and there was a significant increase in the size of canals in the mandible with increasing age. Female mandibles had more porous cortices. No relationship was found between cortical porosity and the degree of bone mineralization.

Combining confocal and BSE SEM imaging for bone block surfaces.

The present report presents a method for the correlation of qualitative and quantitative BSE SEM imaging with confocal scanning light microscopy (CSLM) imaging modes applied to bone samples embedded in PMMA. The SEM has a proper digital scan generator: we leave the BSE image unchanged, and match the CSLM image to it, because the CSLM scan mechanism is not digital, though the signal is digitised. Our overlapping program uses a linear transformation matrix which projects one system to the other, calculated by finding three corresponding points in BSE and CSLM pictures. BSE images are empty where cells and osteoid are present. Fluorescence mode CSLM fills in these gaps. The combination images enhance our understanding of what is going on - and re-establish the need for good cellular preservation.

Ultrastructural study of mineralization of a strontium-containing hydroxyapatite (Sr-HA) cement in vivo.

The purpose of this study was to investigate the mineralization leading to osseointegration of strontium-containing hydroxyapatite (Sr-HA) bioactive bone cement injected into cancellous bone in vivo. Sr-HA cement was injected into the ilium of rabbits for 1, 3, and 6 months. The bone mineralization area was found to be largest at 3 months, then at 1 month, and smallest at 6 months (p < 0.01) measured with tetracycline labeling. Osseointegration of Sr-HA cement was achieved at 3 months as observed by scanning electron microscopy. A high calcium and phosphorus area was observed at the interface of bone-Sr-HA cement determined by energy-dispersive X-ray analysis. Transmission electron microscopy gave evidence of the mechanism of bone formation. Dissolution of Sr-HA into debris by the bone remodeling process was thought to increase the concentration of calcium and phosphorus at the interface of bone-Sr-HA cement and stimulate bone formation. Crystalline Sr-HA formed an amorphous layer and dissolved into the surrounding solution, then apatite crystallites were precipitated and formed new bone at 3 months. This young bone then becomes mature bone, which bonds tightly to the Sr-HA cement with collagen fibers inserted perpendicularly after 6 months.

Design and fabrication of standardized hydroxyapatite scaffolds with a defined macro-architecture by rapid prototyping for bone-tissue-engineering research.

This investigation describes the production and characterization of calcium phosphate scaffolds with defined and reproducible porous macro-architectures and their preliminary in vitro and in vivo bone-tissue-engineered response. Fugitive wax molds were designed and produced using a rapid prototyping technique. An aqueous hydroxyapatite slurry was cast in these molds. After sintering at 1250 degrees C and then cleaning, dimensional and material characterizations of the scaffolds were performed. The resulting scaffolds represented the design, and their dimensions were remarkably consistent. A texture inherent to the layer-by-layer production of the mold was impressed onto the vertical surfaces of the scaffolds. The surface roughness (R(a)) of the textured surfaces was significantly greater than that of the nontextured surfaces. Material analyses revealed a beta-TCP phase in addition to hydroxyapatite for the molded ceramics. Non-molded control ceramics exhibited only hydroxyapatite. Thirty scaffolds were seeded with culture-expanded goat bone-marrow stromal cells (BMSCs) and implanted subcutaneously in nude mice for 4 or 6 weeks. Histology revealed mineralized bone formation in all the scaffolds for both implantation periods. After 4 weeks, bone was present primarily as a layer on scaffold surfaces. After 6 weeks, the surface bone formation was accompanied by bone budding from the surface and occasional bridging of pores. This budding and bridging bone formation almost always was associated with textured scaffold surfaces. However, the area percentage of bone in pores was similar for the 4- and 6-week implantation periods.

Effects of vertebral bone fragility and bone formation rate on the mineralization levels of cancellous bone from white females.

Back-scattered electron microscopy was used to study mineralization levels of human iliac cancellous bone of white females (N = 49). Mineralization levels were assessed by converting bone pixel grayscale levels to atomic number (Z) using known calibration standards. The data set consisted of bone biopsies from normal and vertebral fracture subjects that had either high or low values for bone formation rate (BFR(s)) within their respective groups (fracture/low BFR(s), N = 12; fracture/high BFR(s), N = 10; normal/low BFR(s), N = 12; normal/high BFR(s), N = 15). The following three measures of mineralization were quantitatively determined for each specimen: an overall mean mineralization (Z(mean)), the mineralization of trabecular packets deep within the interior of trabeculae (Z(deep)), and the mineralization of superficial exterior packets (Z(superficial)). Two-way analysis of variance revealed that the high BFR(s) group had a significantly lower Z(superficial) than the low BFR(s) group [mean (SD) 10.383 (0.270) vs. 10.563 (0.289)], and there was no significant interaction. BFR(s) had no effect on Z(mean) or Z(deep). For the pooled data, Z(deep) was significantly higher than Z(superficial) [10.866 (0.242) vs. 10.471 (0.291)]. There was no significant difference in Z(mean), Z(deep), or Z(superficial) between normals and those with vertebral fracture, but the standard deviations of the mineralization measures in the fracture group were at least double that of the normal group. Frequency histograms show that the two groups have fundamentally different mineralization distributions. The normal group demonstrates typical Gaussian distributions centered around the mean, and the distributions of the fracture group are bimodal, with peaks occurring at either the high or low tails of the distributions of the normal group. We hypothesize that both low and high patterns of mineralization might detrimentally affect bone material properties, with low mineralization levels causing reduced stiffness and strength and high mineralization resulting in reduced fracture toughness. The degree to which the mineralization differences may affect strength and stiffness of individual elements is estimated. The higher standard deviations of mineralization measures in the fracture group may reflect an inability to properly regulate trabecular level stress and strain. Forward stepwise regression analysis showed significant relationships between Ob.S/OS and both Z(superficial) and Z(mean), suggesting that the osteoblast may play an important role in regulating mineralization.

Increased bone resorption with decreased activity and increased recruitment of osteoblasts in osteogenesis imperfecta type I.

An iliac bone biopsy from an adult male, 58 years of age, with osteogenesis imperfecta type I was studied by bone histomorphometry after double-fluorescence labeling with tetracycline. Low bone mineral density (BMD) of the radius, measured by dual-energy X-ray absorptiometry (DXA) was associated with high levels of urinary deoxypyridinoline and serum bone-specific alkaline phosphatase and osteocalcin. At the tissue level, low cancellous bone volume (BV/TV) was associated with increased eroded surface (ES/BS) and a relatively increased osteoclast number (N.Oc/BS). Osteoid thickness (O.Th) was also decreased as a result of decreased bone matrix synthesis, in terms of decreased osteoblastic activity. However, osteoid surface (OS/BS) and osteoblast surface (ObS/BS), in terms of the number of osteoblasts, were increased. We conclude that the patient showed cancellous osteopenia, which was likely due to increased bone resorption with decreased activity and increased recruitment of osteoblasts.