RNA-sequencing reveals positional memory of multipotent mesenchymal stromal cells from oral and maxillofacial tissue transcriptomes.

BACKGROUND: Multipotent mesenchymal stromal cells (MSCs) can be isolated from numerous tissues and are attractive candidates for therapeutic clinical applications due to their immunomodulatory and pro-regenerative capacity. Although the minimum criteria for defining MSCs have been defined, their characteristics are known to vary depending on their tissue of origin. RESULTS: We isolated and characterized human MSCs from three different bones (ilium (I-MSCs), maxilla (Mx-MSCs) and mandible (Md-MSCs)) and proceeded with next generation RNA-sequencing. Furthermore, to investigate the gene expression profiles among other cell types, we obtained RNA-seq data of human embryonic stem cells (ESCs) and several types of MSCs (periodontal ligament-derived MSCs, bone marrow-derived MSCs, and ESCs-derived MSCs) from the Sequence Reads Archive and analyzed the transcriptome profile. We found that MSCs derived from tissues of the maxillofacial region, such as the jaw bone and periodontal ligament, were HOX-negative, while those derived from other tissues were HOX-positive. We also identified that MSX1, LHX8, and BARX1, an essential regulator of craniofacial development, were strongly expressed in maxillofacial tissue-derived MSCs. Although MSCs may be divided into two distinct groups, the cells originated from over the neck or not, on the basis of differences in gene expression profile, the expression patterns of all CD antigen genes were similar among different type of MSCs, except for ESCs. CONCLUSIONS: Our findings suggest that MSCs from different anatomical locations, despite meeting general characterization criteria, have remarkable differences in gene expression and positional memory. Although stromal cells from different anatomical sources are generally categorized as MSCs, their differentiation potential and biological functions vary. We suggested that MSCs may retain an original tissue memory about the developmental process, including gene expression profiles. This could have an important impact when choosing an appropriate cell source for regenerative therapy using MSCs.

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.

Differences in the developmental origins of the periosteum may influence bone healing.

BACKGROUND AND OBJECTIVE: The jaw bone, unlike most other bones, is derived from neural crest stem cells, so we hypothesized that it may have different characteristics to bones from other parts of the body, especially in the nature of its periosteum. The periosteum exhibits osteogenic potential and has received considerable attention as a grafting material for the repair of bone and joint defects. MATERIAL AND METHODS: Gene expression profiles of jaw bone and periosteum were evaluated by DNA microarray and real-time polymerase chain reaction. Furthermore, we perforated an area 2 mm in diameter on mouse frontal and parietal bones. Bone regeneration of these calvarial defects was evaluated using microcomputed tomography and histological analysis. RESULTS: The DNA microarray data revealed close homology between the gene expression profiles within the ilium and femur. The gene expression of Wnt-1, SOX10, nestin, and musashi-1 were significantly higher in the jaw bone than in other locations. Microcomputed tomography and histological analysis revealed that the jaw bone had superior bone regenerative abilities than other bones. CONCLUSION: Jaw bone periosteum exhibits a unique gene expression profile that is associated with neural crest cells and has a positive influence on bone regeneration when used as a graft material to repair bone defects. A full investigation of the biological and mechanical properties of jaw bone as an alternative graft material for jaw reconstructive surgery is recommended.

Epithelioid angiosarcoma of the ilium: a case report.

Bone epithelioid angiosarcoma (EA) is rare and characterized by large, mildly to moderately pleomorphic epithelioid cells, with abundant eosinophilic cytoplasm, vesicular nuclei, and prominent nucleoli. The tumors may arise in various locations in bone and the patients may present with unifocal or multifocal osseous disease. We present a unifocal lesion case of EA of the ilium in a 62-year-old woman. A needle biopsy of the ilium was performed and first diagnosed poorly differentiated adenocarcinoma based on CKpan and CK18 immunopositivity. The tumor was treated initially with curettage followed by chemotherapy. The final diagnosis on the surgical specimen was epithelioid angiosarcoma.

Biomechanical effects of insertion location and bone cement augmentation on the anchoring strength of iliac screw.

BACKGROUND: Iliac screw loosening has been a clinical problem in the lumbo-pelvic reconstruction. Although iliac screws are commonly inserted into either upper or lower iliac column, the biomechanical effects of the two fixations and their revision techniques with bone cement remain undetermined. The purpose of this study was to compare the anchoring strengths of the upper and lower iliac screws with and without cement augmentation. METHODS: 5 pairs of formalin fixed cadaveric ilia with the bone mineral density values ranged from 0.82 to 0.97 g/cm(2) were adopted in this study. Using screws with 70-mm length and 7.5-mm diameter, 2 conventional iliac screw fixations and their revision techniques with cement augmentation were sequentially established and tested on the same ilium as follows: upper screw, upper cement screw, lower screw, and lower cement screw. Following 2000 cyclic compressive loading of -300 N to -100 N to the screw on a material testing machine, the maximum pull-out strengths were measured and analyzed. FINDINGS: The average pull-out strengths of upper, upper cement, lower, and lower cement screws were 964 N, 1462 N, 1537 N, and 1964 N, respectively. The lower screw showed significantly higher pull-out strength than the upper one (P=0.008). The cement augmentation notably increased the pull-out strengths of both upper and lower screws. The positive correlation between pull-out strength and bone mineral density value was obtained for the 4 fixations. INTERPRETATION: The lower iliac screw technique should be the preferred choice in lumbo-pelvic stabilization surgery; cement augmentation may serve as a useful salvage technique for iliac screw loosening; preoperative evaluation of bone quality is crucial for predicting fixation strength of iliac screw.

Bone material quality in transiliac bone biopsies of postmenopausal osteoporotic women after 3 years of strontium ranelate treatment.

Strontium ranelate (SrR) is a relatively new treatment for osteoporosis. In this study we investigated its potential impact on human bone material quality in transiliac bone biopsies from postmenopausal osteoporotic women treated 3 years with calcium and vitamin D plus either 2 g SrR per day or placebo. Bone mineralization density distribution (BMDD), strontium (Sr) concentration, collagen cross-link ratio, and indentation modulus were analyzed by quantitative backscattered electron imaging, electron-induced X-ray fluorescence analysis, synchrotron radiation induced micro X-ray fluorescence elemental mapping, Fourier transform infrared imaging, and nanoindentation, respectively. The BMDD of SrR-treated patients was shifted to higher atomic numbers (Z(mean) +1.5%, p < .05 versus placebo). We observed Sr being preferentially incorporated in bone packets formed during SrR treatment up to 6% atom fraction [Sr/(Sr + Ca)] depending on the SrR serum levels of the individuals (correlation r = 0.84, p = .018). Collagen cross-link ratio was preserved in SR-treated bone. The indentation modulus was significantly decreased in younger versus older bone packets for both placebo- (-20.5%, p < .0001) and SrR-treated individuals (-24.3%, p < .001), whereas no differences were found between the treatment groups. In conclusion, our findings indicate that after SrR treatment, Sr is heterogeneously distributed in bone and preferentially present in bone packets formed during treatment. The effect of SrR on BMDD seems to be due mainly to the uptake of Sr and not to changes in bone calcium content. Taken together, these data provide evidence that the investigated bone quality determinants at tissue level were preserved in postmenopausal osteoporotic women after 3-year treatment with 2 g SrR per day plus calcium and vitamin D.

Strontium is incorporated into mineral crystals only in newly formed bone during strontium ranelate treatment.

Strontium ranelate has been shown to increase bone mass in postmenopausal osteoporosis patients and to reduce fracture risk. The aim of this study was to investigate the potential influence of strontium ranelate (Protelos) treatment on human bone tissue characteristics and quality at the micro- and nanostructural levels. We investigated transiliac biopsies from patients treated for 36 months with strontium ranelate or placebo (n = 5 per group) using synchrotron radiation with a microbeam combining scanning small-angle scattering, X-ray diffraction, and fluorescence spectroscopy (SAXS/XRD/XRF) for a detailed characterization of the mineral crystals within the collagenous bone matrix. A scanning procedure allowed the simultaneous determination of maps of the chemical composition together with thickness, length, and lattice spacing of these mineral crystals within each of the 15- or 25-microm-wide pixels in a thin bone section. The fluorescence results show that only bone packets or osteons formed during the strontium ranelate treatment contain significant amounts of strontium and that up to 0.5 of 10 calcium atoms in the mineral crystals are replaced by strontium, as revealed by a corresponding shift in apatite lattice spacing. The thickness and length of the plate-shaped bone mineral crystals were not affected by the strontium ranelate treatment. As a consequence, there was no indication for a change in human bone tissue quality at the nanoscale after a 36-month treatment of postmenopausal osteoporotic women with strontium ranelate, except for a partial replacement of calcium by strontium ions in the hydroxyapatite crystals, only in newly formed bone.

Intraoperative adjunctive stem cell treatment in patients with critical limb ischemia using a novel point-of-care device.

INTRODUCTION: In a prospective trial we tested whether adjunctive intraoperative stem cell treatment in patients with critical limb ischemia (CLI) can be performed safely in combination with bypass surgery and/or interventional treatment. The end point of our study was the safety and integrity of a novel point-of-care system used in patients with CLI. METHODS: We included only patients with CLI and tissue loss according to Rutherford categories 4-6. The Harvest Bone Marrow Aspirate Concentrate System consists of an automated, microprocessor-controlled dedicated centrifuge with decanting capability and the accessory BMAC Pack for processing a patient's bone marrow aspirate (BMA). The centrifuge is portable and enables BMA to be rapidly processed in the operating room to provide an autologous concentrate of nucleated cells for immediate injection. The surgeon aspirated 120 ml BMA from the iliac crest. RESULTS: Eight consecutive patients were treated according to the study protocol. The mean follow-up period was 9.2 months (range 2-18). Stem cells were always injected during the final revascularization attempt. One minor amputation and two major amputations were required. In five of eight patients there was a discrete increase in the ankle-brachial index post-stem cell treatment. The dose of stem cells after centrifugation was 17.2 (range 13.8-54.2)x10E6 CD34-positive cells and 7.8 (range 1.8-35.9)x10E6 CD133-positive cells. The injected dose of VEGFR-2-coexpressing stem cells was 0.5-5.7x10E4. CONCLUSION: We were able to show that the buffy coat preparation using a point-of-care system is a simple and fast method to enrich stem cells from BMAs. This automated system gives high recovery rates and good reproducibility.

Natural bone collagen scaffold combined with autologous enriched bone marrow cells for induction of osteogenesis in an ovine spinal fusion model.

Autologous bone graft, the standard of bone grafting in achieving spinal fusion, is associated with several limitations and complications. The use of bone marrow cells (BMCs) as a potential cell source for spinal fusion, combined with a suitable scaffold to promote bone formation, may be a better choice. The aims of this study were to evaluate the efficacy of natural bone collagen scaffold (NBCS) combined with autologous-enriched BMCs for induction of osteogenesis in vitro and in vivo. Ovine-enriched BMCs were co-cultured with NBCS for 1, 2, 3, and 4 weeks to investigate whether NBCS would support the population expansion and differentiation of enriched BMCs. Using an ovine interbody fusion model, NBCS seeded with autologous enriched BMCs was implanted into the lumbar disc space. Fusion outcomes were compared with the use of the autograft, NBCS without BMCs, and BMCs without NBCS. In vitro results demonstrated that NBCS facilitated the population expansion and differentiation of ovine-enriched BMCs, promoting the expression of collagen type I and the formation of a mineralized matrix. The use of NBCS combined with enriched BMCs in vivo enhanced the spinal fusion rate (6 of 6 at 10 week) (p < 0.05), the biomechanical stiffness of fusion masses, and bone volume at the fusion site (p < 0.05). Histological findings also revealed that a combination of NBCS and BMCs induced new bone formation that integrated well with host bone tissue. In conclusion, NBCS is an effective scaffold that supports ovine-enriched BMCs. The combination of NBCS and BMCs may be a useful alternative for autograft in induction of spinal fusion.

RANKL is a mediator of bone resorption in idiopathic hypercalciuria.

BACKGROUND AND OBJECTIVES: This study aimed to determine the expression of osteoprotegerin, receptor activator of nuclear factor kappaB ligand, interleukin-1alpha, transforming growth factor-beta, and basic fibroblast growth factor in stone-forming patients with idiopathic hypercalciuria. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Immunohistochemical analysis was performed in undecalcified bone samples previously obtained from 36 transiliac bone biopsies of patients who had idiopathic hypercalciuria and whose histomorphometry had shown lower bone volume, increased bone resorption, and prolonged mineralization lag time. RESULTS: Bone expression of receptor activator of nuclear factor kappaB ligand and osteoprotegerin was significantly higher in patients with idiopathic hypercalciuria versus control subjects. Transforming growth factor-beta immunostaining was lower in patients with idiopathic hypercalciuria than in control subjects and correlated directly with mineralization surface. Interleukin-1alpha and basic fibroblast growth factor staining did not differ between groups. Receptor activator of nuclear factor kappaB ligand bone expression was significantly higher in patients who had idiopathic hypercalciuria and exhibited higher versus normal bone resorption. CONCLUSION: A higher expression of receptor activator of nuclear factor kappaB ligand in bone tissue suggests that increased bone resorption in patients with idiopathic hypercalciuria is mediated by receptor activator of nuclear factor kappaB ligand. Osteoprotegerin bone expression might have been secondarily increased in an attempt to counteract the actions of receptor activator of nuclear factor kappaB ligand. The low bone expression of transforming growth factor-beta could contribute to the delayed mineralization found in such patients.

Quantitative assessment of growth factors in reaming aspirate, iliac crest, and platelet preparation.

Large bony defects and non-unions are still a complication in trauma and orthopedic surgery. Treatment strategies include the use of autogenous materials (iliac crest), allogenic bone, bone substitutes, and currently stimulation with growth factors such as BMP-2, BMP-7 or the growth factors containing platelet-rich plasma (PRP). Another source of bone graft material might be the cuttings produced during intramedullary reaming. The aim of this study was to compare the quantity of various growth factors found within iliac crest, bony reaming debris, reaming irrigation fluid, and platelet-rich plasma. Iliac crest and reaming debris and irrigation samples were harvested during surgery. PRP was prepared from blood. The growth factors in the bony materials (iliac crest or reaming debris) and of the liquid materials (platelet-poor plasma (PPP), platelet-rich plasma (PRP) or reaming irrigation) were compared. Elevated levels of FGFa, PDGF, IGF-I, TGF-beta1 and BMP-2 were measured in the reaming debris as compared to iliac crest curettings. However, VEGF and FGFb were significantly lower in the reaming debris than from iliac crest samples. In comparing PRP and PPP all detectable growth factors, except IGF-I, were enhanced in the platelet-rich plasma. In the reaming irrigation FGFa (no measurable value in the PRP) and FGFb were higher, but VEGF, PDGF, IGF-I, TGF-beta1 and BMP-2 were lower compared to PRP. BMP-4 was not measurable in any sample. The bony reaming debris is a rich source of growth factors with a content comparable to that from iliac crest. The irrigation fluid from the reaming also contains growth factors.

Comparison of mineral quality and quantity in iliac crest biopsies from high- and low-turnover osteoporosis: an FT-IR microspectroscopic investigation.

Fourier-transform infrared microspectroscopy (FTIRM) allows analysis of mineral content, mineral crystal maturity and mineral composition at approximately 10-micron spatial resolution. Previous FTIRM analyses comparing 4-micron thick sections from non-decalcified iliac crest biopsies from women with post-menopausal osteoporosis, as contrasted with iliac crest tissue from individuals without evidence of metabolic bone disease, demonstrated significant differences in average mineral content (decreased in osteoporosis) and mineral crystal size/perfection (increased in osteoporosis). More importantly, these parameters, which vary throughout the tissue in relation to the tissue age in healthy bone, showed no such variation in bone biopsies from patients with osteoporosis. The present study compares the spatial and temporal variation in mineral quantity and properties in trabecular bone in high- and low-turnover osteoporosis. Specifically, six biopsies from women (n=5) and one man with high-turnover osteoporosis (age range 39-77) and four women and two men with low turnover osteoporosis (age range 37-63) were compared to ten "normal" biopsies from three men and seven woman (age range: 27-69). "High turnover" was defined as the presence of increased resorptive surface, higher than normal numbers of osteoclasts and greater than or equal to normal osteoblastic activity. "Low turnover" was defined as lower than normal resorptive surface, decreased osteoclast number and less than normal osteoblastic activity. Comparing variations in FTIR-derived values for each of the parameters measured at the surfaces of the trabecular bone to the maximum value observed in multiple trabeculae from each person, the high-turnover samples showed little change in the mineral: matrix ratio, carbonate: amide I ratio, crystallinity and acid phosphate content. The low-turnover samples also showed little change in these parameters, but in contrast to the high-turnover samples, the low-turnover samples showed a slight increase in these parameters, indicative of retarded, but existent resorption and formation. These data indicate that FTIR microspectroscopy can provide quantitative information on mineral changes in osteoporosis that are consistent with proposed mechanisms of bone loss.

Semiquantitative mRNA measurements of osteoinductive growth factors in human iliac-crest bone: expression of LMP splice variants in human bone.

Although osteotropic growth factors are known to play an important role in bone metabolism, knowledge about their expression in relation to age, sex and smoking remains limited. In this study we report mRNA levels of the recently discovered Lim mineralization protein splice variants (LMP-1, LMP-2, LMP-3) and the established osteotropic growth factors BMP-2, BMP-6, BMP-7, TGF-beta, IGF-I, IGF-II and b-FGF in human iliac crest bone. Standardized bone biopsy specimens were obtained from the iliac crest during graft harvesting in 62 patients (38 males, 24 females, mean age 44.7 years, range 13-78 years) undergoing spinal surgery. Samples were immediately stored in liquid nitrogen for PCR analysis. Semi-quantitative RT-PCR was performed for TGF-beta, IGF-I, IGF-II, BMP2, BMP-6, BMP7, bFGF, LMP-1, LMP-2 and LMP-3 using beta-actin as internal standard. Triplicate measurements were made of each growth factor and beta-actin. mRNA for all examined growth factors was detected in 69% of the specimens. The lowest degree of detection was present for b-FGF and BMP-2, both of which were found in 85% of the specimens. LMP-1 was detected in 98% of the specimens. LMP-2 in 94% and LMP-3 in 27%, respectively. LMP-1 was generally expressed in higher amounts than LMP-2 and LMP-3. Nondetectable levels of the growth factors were more frequent in the >60-year-old males compared with >60-year-old females ( P < 0.05) and <60-year-old males ( P < 0.01). LMP-1 expression was more variable among young individuals, but mean values were similar between age groups. TGF-beta, BMP-2 and BMP-7 values did not differ between age groups, but generally a higher variation was found among older patients. IGF-I values were significantly higher ( P < 0.05) in males over 60 years, whereas the highest level of bFGF mRNA was present in males younger than 20 years ( P < 0.05). In addition, regression analysis revealed correlation between BMP-2 and BMP-7 (R2 = 0.74, P < 0.0005), LMP-2 and BMP-2 (R2 = 0.27, P < 0.0005) and LMP-2 and bFGF (R2 = 0.40, P < 0.0005). In conclusion, we have demonstrated expression of LMP-1 and LMP-2 in human bone. LMP-1 was expressed in higher amounts and showed a higher degree of variation among young individuals. LMP-2 was correlated to a number of other growth factors, suggesting that LMPs may also play a role in human bone metabolism. Higher variation in the expression of TGF-beta, BMP-2 and BMP-7 was found in the older age groups, but whether or not this can be correlated to age-related changes in bone turnover requires further studies.

[Effect of strontium on bone metabolism in hemodialysis patients]. / Efecto del estroncio sobre el metabolismo óseo en pacientes en hemodiálisis.

The objective of this study was to assess the relationship between the bone strontium content and bone histomorphometric parameters in bone biopsies from patients with chronic renal failure undergoing hemodialysis. The study was carried out in 74 illiac crest bone biopsies from patients with renal osteodystrophy from different worldwide regions (Argentina, Portugal and Spain). They were underwent to histological and histomorphometric evaluation. The bone strontium/calcium ratio was measured by quadrupole inductively coupled plasma-mass spectrometry. The samples were classified into groups according to histological criteria: hyperparathyroidism (HP), mixed (MX), osteomalacia (OM) and adynamic bone disease (ABD). Serum PTH and alkaline phosphatase before biopsy were available in most of the patients. No correlation was found between the different histomorphometric parameters and the Sr/Ca ratio. The one way ANOVA test showed statistical differences in the Sr/Ca ratio of the different histological forms (HP: 0.58 +/- 0.39; MX: 1.16 +/- 0.74; OM: 1.10 +/- 0.46; ABD: 0.91 +/- 0.40 microgram Sr/mg Ca; p < 0.003). The post-Hoc analysis showed differences between HP and MX. The biopsies having greater or equal values than 1.4 micrograms Sr/mg Ca showed higher levels of bone formation histomorphometric parameters and serum alkaline phosphatase (395 +/- 519 vs 1,022 +/- 989 UI/L, p < 0.05). Although it has been found that the biopsies with higher bone strontium had higher levels of osteoid tissue (characteristic of osteomalacia), the hypothesis of strontium-induced osteomalacia could not be demonstrated.

Quantification of the degree of mineralization of bone in three dimensions using synchrotron radiation microtomography.

The availability of three-dimensional measuring techniques coupled to specific image processing methods opens new opportunities for the analysis of bone structure. In particular, synchrotron radiation microtomography may provide three-dimensional images with spatial resolution as high as one micrometer. Moreover, the use of a monoenergetic synchrotron beam, which avoids beam-hardening effects, allows quantitative measurements of the degree of mineralization in bone samples. Indeed, the reconstructed gray levels of tomographic images correspond directly to a map of the linear attenuation coefficient within the sample. Since the absorption depends on the amount of mineral content, we proposed a calibration method to evaluate the three-dimensional distribution of the degree of mineralization within the sample. First a theoretical linear relationship modeling the linear attenuation coefficient as a function of the hydroxyapatite concentrations was derived. Then, an experimental validation on phantoms confirmed both the accuracy of the image processing tools and the experimental setup used. Finally, the analysis of the degree of mineralization in four iliac crest bone biopsy samples was reported. Our method was compared to the reference microradiography technique, currently used for this quantification in two dimensions. The concentration values of the degree of mineralization were found with both techniques in the range 0.5-1.6 g of mineral per cubic centimeter of bone, both in cortical and in trabecular region. The mean difference between the two techniques was around 4.7%, and was slightly higher in trabecular region than in cortical bone.

Determination of trace elements and calcium in bone of the human iliac crest by atomic absorption spectrometry.

A rapid and reliable analytical method for the determination of trace elements in human bone by atomic absorption spectrometry is reported. Calcium was determined to estimate the homogeneity of samples. Human bone from the iliac crest was obtained at autopsy of adult subjects. Before analysis samples were decomposed by microwave digestion and acid digestion in a Parr bomb. Zinc, rubidium, strontium, calcium and iron were determined by flame atomic absorption spectrometry (FAAS) and aluminium, copper and lead by electrothermal atomic absorption spectrometry (ETAAS) at optimum measurement conditions. The results for the two digestion procedures agreed for zinc, rubidium and calcium within +/-5%, for copper within +/-7% and for strontium, iron, aluminium and lead within +/-10%. The repeatability of measurement (R.S.D.) for determination of calcium and trace elements after microwave digestion and acid digestion in a Parr bomb was tested in one representative autopsy bone sample by six parallel determinations. It was found to be better than +/-5% either for microwave digested samples or samples digested in a Parr bomb, for all elements determined by FAAS and ETAAS techniques. The accuracy of the applied digestion procedures was checked by analysis of trace elements in NIST SRM 1486 Bone Meal reference material. Good agreement of the results with certified values was obtained for both digestion procedures. The microwave procedure developed for digestion of small amounts of sample was applied in trace elements analysis of bone biopsy samples from dialysis patients.

Validation of quantitative backscattered electron imaging for the measurement of mineral density distribution in human bone biopsies.

The measurement of bone mineral density (BMD) using X-rays is usually employed to monitor the mineral content in a given portion of bone. However, this method cannot differentiate between changes in bone volume or in degree of mineralization of the bone matrix. In contrast to BMD, bone mineral density distribution (BMDD), as measured on bone sections by quantitative backscattered electron imaging (qBEI), is able to distinguish differences in the degree of mineralization. For routine clinical research, we have validated the method of calibration and standardization of the backscattered electron (BE) signal. Carbon and aluminum were used as reference materials for BE gray levels and osteoid and apatite for calcium concentration. Experiments were performed to get knowledge about precision (intraassay variance-instrumental stability and interassay variance-reproducibility) and accuracy (standardization) of this method as well as the biological variance (intraindividual and interindividual) in human bone. On transiliac biopsies or necropsies from 20 individuals having had accidental death (13 females, 7 males, age 30-85 years) BMDD measurements were conducted. The patients' medical history as well as the histomorphology of these bones showed no evidence of metabolic bone disease. For instance, the standard deviations of the weighted mean calcium concentrations were <0.3%, <0.4%, <0.9%, and <2.6% of the mean for the intraassay, interassay, intraindividual, and interindividual variations, respectively. In addition, a mean BMDD histogram for transiliac bone specimens was calculated from the 20 aforementioned individuals. The method used allows detection of the degree of mineralization independently from the actual bone volume, a result that seems to be of special interest in the assessment of the effect of treatments for osteoporosis. The power of this technique is demonstrated by using bone from a patient with a metabolic bone disease. In this case of osteomalacia due to celiac disease, the mean calcium concentration in the bone matrix was reduced by 19.3% as compared with normal.

FTIR microspectroscopic analysis of normal human cortical and trabecular bone.

Fourier transform infrared microspectroscopy (FTIRM) has been used to study the changes in mineral and matrix content and composition in replicate biopsies of nonosteoporotic human cortical and trabecular bone. Changes in osteonal bone in these same samples were reported previously. Spectral maps along and across the lamellae were obtained from iliac crest biopsies of two necropsy cases. Mineral:matrix ratios, calculated from the integrated areas of the phosphate nu1, nu3 band at 900-1200 cm-1 and the amide I band at approximately 1585-1725 cm-1, respectively, were relatively constant in both directions of analysis, i.e., along and across the lamellae. Analysis of the components of the nu1, nu3 phosphate band with a combination of second-derivative spectroscopy and curve fitting revealed the presence of 11 major underlying moieties. Of these, the ratio of the relative areas of the two underlying bands at approximately 1020 and approximately 1030 cm-1 has been shown to be a sensitive index of variation in crystal perfection in both human osteonal bone and in synthetic, poorly crystalline apatites. This ratio was calculated in both cortical and trabecular bone from human iliac crest biopsies along and across the lamellae. The ratio decreased, going from the periosteum to the medullary cavity in the cortical bone, and from the periphery towards the center of trabeculae. These observations were consistent within serial sections obtained from the same biopsy, multiple biopsies obtained from the same necropsy specimen, and biopsies obtained from the two different necropsy specimens. The results presented here along with previously reported changes in osteonal bone show a relation between bone age and "crystallinity/maturity" (a parameter dependent on crystallite size, hydroxyapatite-like stoichiometry, abundance of substituting ions such as CO32-; the more crystalline/mature, the more hydroxyapatite-like stoichiometry, the bigger the crystallite size, the less the ion substitution by ions such as CO32-) as deduced by the 1020/1030 cm-1 ratio. Invariably, younger normal bone is less mature/crystalline than older. These results provide a "baseline" for description of mineral properties, to which diseased bones may be compared.

Bony content of oxalate in patients with primary hyperoxaluria or oxalosis-unrelated renal failure.

Oxalate retention occurs in end-stage renal failure. Regular dialysis treatment does not prevent progressive accumulation of oxalate in cases of ESRF due to primary hyperoxaluria (PH), whereas such accumulation seldom seems to occur in oxalosis-unrelated ESRF. To elucidate this issue we have measured the bony content of oxalate on biopsies of the iliac crest taken from 32 uremic patients, 7 of them with ESRF associated with PH1 (6 cases) or PH2 (1 case). Ten subjects with normal renal function and no evidence of metabolic bone disease were taken as controls. Only trace amounts levels of oxalate were detected in normal subjects and oxalate to phosphate ratio was below 3:10,000. Non-PH dialyzed patients exhibited fivefold increases in oxalate levels, which rose to 5.1 +/- 3.6 mumol/g bony tissue. Calcium oxalate was estimated to represent 0.18% of the hydroxyapatite content of bone. Oxalate amounts were neither related to pre-dialysis plasma levels of oxalate, nor with duration of dialysis treatment, suggesting that accumulation was not progressive disorder. Oxalate levels were slightly higher in patients with a low turnover osteodystrophy compared to those with a high turnover pattern. Dialyzed patients with PH had remarkable increases in oxalate levels, which ranged between 14.8 and 907 mumol/g bony tissue. Oxalate deposition appeared to be progressive in that oxalate levels were significantly related to time on dialysis. In three patients calcium oxalate was a significant fraction of the mineralized bone. The occurrence of calcium oxalate crystals affected the histomorphometric patterns, that were featured by an increase in resorptive areas and a decrease in bone formation rate.(ABSTRACT TRUNCATED AT 250 WORDS)