Environmental fungi and bacteria facilitate lecithin decomposition and the transformation of phosphorus to apatite.

Organophosphorus compounds (OP) are stable P source in nature, and can increase eutrophication risk in waterbodies. Lecithin was the most difficult OP to be broken down. In this study, two typical phosphate-solubilizing microorganisms, Aspergillus niger and Acinetobacter sp., were applied to evaluate their ability to decompose both inorganic phosphates and lecithin. A. niger and Acinetobacter sp. could solubilize calcium phosphates by secreting various organic acids, e.g., oxalic and formic acids. The fungus, A. niger, shows significantly higher ability of solubilizing these inorganic phosphates than Acinetobacter sp., primarily due to its secretion of abundant oxalic acid. However, the bacterium, Acinetobacter sp., could secrete more acid phosphatase than A. niger for lecithin decomposition, i.e., 9300 vs. 8500 µmol L-1 h-1. Moreover, after addition of CaCl2, the released P from lecithin was transformed to stable chlorapatite in the medium. To the contrast, Ca cations inclined to form calcium oxalate (rather than stable phosphate mineral) after the incubation of A. niger, as it induced relatively acidic environment after breaking down lecithin. Therefore, this work sheds light on the bright future of applying bacteria and Ca cations in OP pollutant management.

Delicate changes of bioapatite mineral in pig femur with addition of dietary xylooligosaccharide: Evidences from Raman spectroscopy and ICP.

Bone mineral is strongly correlated with performance and health of animal bodies. The mineral bioapatite (BAp) is the dominant component in bone tissue. This study investigated mineralogical changes of BAp in pig femur by Raman spectroscopy and inductively coupled plasma optical emission spectrometry (ICP-OES). The pigs had been raised with various xylooligosaccharide (XOS) additions at two stages of growth (growing and fattening periods). The results show that XOS can decrease the degree of carbonate substitution for PO4 in BAp mineral and improve the mineral's crystallinity. ICP data is consistent with the Raman results, that is the low solubility of bone BAp for pigs fed with XOS. Additionally, the effect of XOS is much better in the growing period (before 65 kg) than in the fattening period (after 65 kg). Moreover, the high addition of XOS (within the range of 0.1-0.5 g/kg) would be appropriate to improve the crystallinity of bone BAp. This study sheds light on applying Raman and ICP techniques to investigate the delicate changes of mineral in pig bones undergoing different managements.

Effect of ceramic calcium-phosphorus ratio on chondrocyte-mediated biosynthesis and mineralization.

The osteochondral interface functions as a structural barrier between cartilage and bone, maintaining tissue integrity postinjury and during homeostasis. Regeneration of this calcified cartilage region is thus essential for integrative cartilage healing, and hydrogel-ceramic composite scaffolds have been explored for calcified cartilage formation. The objective of this study is to test the hypothesis that Ca/P ratio of the ceramic phase of the composite scaffold regulates chondrocyte biosynthesis and mineralization potential. Specifically, the response of deep zone chondrocytes to two bioactive ceramics with different calcium-phosphorus ratios (1.35 ± 0.01 and 1.41 ± 0.02) was evaluated in agarose hydrogel scaffolds over two weeks in vitro. It was observed that the ceramic with higher calcium-phosphorus ratio enhanced chondrocyte proliferation, glycosaminoglycan production, and induced an early onset of alkaline phosphorus activity, while the ceramic with lower calcium-phosphorus ratio performed similarly to the ceramic-free control. These results underscore the importance of ceramic bioactivity in directing chondrocyte response, and demonstrate that Ca/P ratio is a key parameter to be considered in osteochondral scaffold design. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2694-2702, 2017.

Calcium and vitamin D have a synergistic role in a rat model of kidney stone disease.

Vitamin D supplementation in humans should be accompanied by calcium administration to avoid bone demineralization through vitamin D receptor signaling. Here we analyzed whether long-term exposure of rats to vitamin D supplementation, with or without a calcium-rich diet, would promote kidney stone formation. Four groups of rats received vitamin D alone (100,000 UI/kg/3 weeks), a calcium-enriched diet alone, both vitamin D supplementation and calcium-enriched diet, or a standard diet (controls) for 6 months. Serum and urine parameters and crystalluria were monitored. Kidney stones were assessed by 3-dimensional micro-computed tomography, infrared spectroscopy, von Kossa/Yasue staining, and field emission scanning electron microscopy. Although serum calcium levels were similar in the 4 groups, rats receiving vitamin D had a progressive increase in urinary calcium excretion over time, especially those receiving both calcium and vitamin D. However, oral calcium supplementation alone did not increase urinary calcium excretion. At 6 months, rats exposed to both calcium and vitamin D, but not rats exposed to calcium or vitamin D alone, developed significant apatite kidney calcifications (mean volume, 0.121 mm(3)). Thus, coadministration of vitamin D and increased calcium intake had a synergistic role in tubular calcifications or kidney stone formation in this rat model. Hence, one should be cautious about the cumulative risk of kidney stone formation in humans when exposed to both vitamin D supplementation and high calcium intake.

The effect of calcium and vitamin D supplementation on osteoporotic rabbit bones studied by vibrational spectroscopy.

Fourier transform infrared spectroscopy is utilized to examine the effects of increased calcium, vitamin D, and combined calcium-vitamin D supplementation on osteoporotic rabbit bones with induced inflammation. The study includes different bone sites (femur, tibia, humerus, vertebral rib) in an effort to explore possible differences among the sites. We evaluate the following parameters: mineral-to-matrix ratio, carbonate content, and non-apatitic species (labile acid phosphate and labile carbonate) contribution to bone mineral. Results show that a relatively high dose of calcium or calcium with vitamin D supplementation increases the bone mineralization index significantly. On the other hand, vitamin D alone is not as effective in promoting mineralization even with high intake. Mature B-type apatite was detected for the group with calcium supplementation similar to that of aged bone. High vitamin D intake led to increased labile species concentration revealing bone formation. This is directly associated with the suppression of pro-inflammatory cytokines linked to induced inflammation. The latter is known to adversely alter bone metabolism, contributing to the aetiopathogenesis of osteoporosis. Thus, a high intake of vitamin D under inflammation-induced osteoporosis does not promote mineralization but suppresses bone resorption and restores metabolic balance.

Biochemical and molecular aspects of spectral diagnosis in calcinosis cutis.

Calcinosis cutis (CC) is a type of calcinosis wherein insoluble compounds or salts deposited on the skin. Clinical diagnosis of CC is usually achieved through time consuming histopathological or immunohistochemical procedures, but it can only be empirically identified by experienced practitioners. The use of advanced vibrational spectroscopy has been recently shown to have great potential as a diagnostic technique for various diseased tissues because it analyses the chemical composition of diseased tissue rather than its anatomy and predicts disease progression. This review article includes a summary of the application of Fourier transform infrared (FT-IR) and Raman spectroscopic or microspectroscopic analysis for the rapid diagnosis and identification of the chemical composition of skin calcified deposits in patients with various CC symptoms. Both advanced techniques not only can detect the types of insoluble salts such as calcium phosphate, calcium carbonate, and monosodium urate, and ß-carotene in the calcified deposits of human skin tissue but also can directly differentiate the carbonate substitution in the apatite structure of the skin calcified deposits. In particular, the combination of both vibrational techniques may provide complementary information to simultaneously assess the intact components of the calcified deposits. In the future, both FT-IR and Raman vibrational microspectroscopic techniques will become available tools to support the standard test techniques currently used in some clinical diagnoses. Molecular spectroscopy technique is rapidly changing disease diagnosis and management.

A new technique for the assessment of the 3D spatial distribution of the calcium/phosphorus ratio in bone apatite.

The value and distribution of calcium/phosphorus (Ca/P) ratio in bone vary between healthy and osteoporotic bone. The purpose of this study was the development of a technique for the assessment of the 3D spatial distribution of Ca/P ratio in bone apatite, which could eventually be implemented through a conventional computed tomography (CT) system. A three-material mass-fraction decomposition CT dual energy analysis was optimized. The technique was validated using ten bone phantoms of different, known Ca/P ratio. Their measured average Ca/P ratio showed a mean/maximum deviation from the expected Ca/P ratio of 0.24/0.35. Additionally, three healthy and three inflammation-mediated osteoporotic (IMO) collagen-free rabbit tibia bone samples were assessed, providing promising preliminary results on real bone tissue. The average Ca/P ratios in all IMO samples (1.64-1.65) were found to be lower than in healthy samples (1.67-1.68). Osteoporotic regions in IMO samples were located using Ca/P ratio colour maps and Ca/P ratio values as low as 1.40 ± 0.26 were found. The low Ca/P ratio volume proportion in IMO samples (12.8%-13.9%) was found to be higher than in healthy (5.8%-8.3%) samples. A region growing technique showed a higher homogeneity of Ca/P ratio in healthy than in IMO bone samples.

Microbial processing of apatite rich low grade Indian uranium ore in bioreactor.

Bioreactor leaching using enriched culture of Acidithiobacillus ferrooxidans and Leptosprillium ferrooxidans was investigated for the apatite rich Indian (Narwapahar) uranium ore. Bioreactor leaching of Narwapahar ore of <45 µm size at pH 2.0 and 10% (w/v) PD using 10% (v/v) inoculum of the bacterium at 35 °C (A. ferrooxidans) and 40 °C (L. ferrooxidans), solubilised 57% and 63% uranium in 5 days, respectively; the E(SCE) values being 561 and 588 mV. Leaching kinetics improved so much so that ~83% uranium was recovered in just 10h with 10% inoculum of A. ferrooxidans containing biogenic Fe(3+); at 20% PD uranium recovery rose to 87%. Role of temperature (25-40 °C) was noticed with 90.3% uranium bioleaching in 10h at 40 °C with L. ferrooxidans as against 77% leaching with A. ferrooxidans at pH 2.0, 40 °C and 20% (w/v) PD.

Plant-driven weathering of apatite--the role of an ectomycorrhizal fungus.

Ectomycorrhizal (EcM) fungi are increasingly recognized as important agents of mineral weathering and soil development, with far-reaching impacts on biogeochemical cycles. Because EcM fungi live in a symbiotic relationship with trees and in close contact with bacteria and archaea, it is difficult to distinguish between the weathering effects of the fungus, host tree and other micro-organisms. Here, we quantified mineral weathering by the fungus Paxillus involutus, growing in symbiosis with Pinus sylvestris under sterile conditions. The mycorrhizal trees were grown in specially designed sterile microcosms in which the supply of soluble phosphorus (P) in the bulk media was varied and grains of the calcium phosphate mineral apatite mixed with quartz, or quartz alone, were provided in plastic wells that were only accessed by their fungal partner. Under P limitation, pulse labelling of plants with (14)CO(2) revealed plant-to-fungus allocation of photosynthates, with 17 times more (14)C transferred into the apatite wells compared with wells with only quartz. Fungal colonization increased the release of P from apatite by almost a factor of three, from 7.5 (±1.1) × 10(-10) mol m(-2) s(-1) to 2.2 (±0.52) × 10(-9) mol m(-2) s(-1). On increasing the P supply in the microcosms from no added P, through apatite alone, to both apatite and orthophosphate, the proportion of biomass in roots progressively increased at the expense of the fungus. These three observations, (i) proportionately more plant energy investment in the fungal partner under P limitation, (ii) preferential fungal transport of photosynthate-derived carbon towards patches of apatite grains and (iii) fungal enhancement of weathering rate, reveal the tightly coupled plant-fungal interactions underpinning enhanced EcM weathering of apatite and its utilization as P source.

Long-term effect of apatite on ectomycorrhizal growth and community structure.

Ectomycorrhizal (ECM) fungi are efficient at taking up phosphorus (P) from mineral sources, such as apatite, which are not easily available to the host trees. Since ECM fungal species differ in P uptake rates, it can be expected that the composition of the ECM fungal community will change upon exposure to apatite, provided that the P transfer is rewarded by more carbon being transferred to the fungal symbiont. Control and apatite-amended mesh bags were buried in pairs in the humus layer of a P-poor Norway spruce forest. The ECM fungal community that colonized these bags was analyzed by DNA extraction, PCR amplification of the internal transcribed spacer (ITS) region, cloning, and random sequencing. Fungal biomass was estimated by ergosterol analysis. No change in the ECM fungal community structure was seen after 5 years of apatite exposure, although the fungal biomass increased threefold upon apatite amendment. Our results indicate that host trees enhance carbon allocation to ECM fungi colonizing P sources in P-poor forests but the lack of change in the composition of the ECM fungal community suggests that P transfer rates were similar among the species. Alternatively, higher P transfer among certain species was not rewarded with higher carbon transfer from the host.

Pea roots affect immobilisation and solubilisation of phosphorus depending on genotype, stage and phosphorous source.

To assess the efficiency of pea roots to mobilize available phosphorus (P) from P compounds we subjected various pea genotypes to a post-treatment method. Axenic seedlings were raised on P-deficient semisolid synthetic medium using control blanks without a plant otherwise treated in the same way. AlPO(4), CaHPO(4), FePO(4), apatite and meat-bone-meal (MBM) were tested. A genotype was tested from 1-day through 15-days of growth. There were differences between the compounds (p < 0.001). P was dissolved from CaHPO(4) with apparent maxima at 72-h intervals and to a significantly lesser extent from MBM. With AlPO(4), FePO(4) and apatite, the roots did not show a dissolving effect, but, on the contrary, significantly immobilised P. In each case a correlation with an increase in acidity, H(+) (p < 0.001) was observed. The correlation was negative in the AlPO(4), FePO(4) and apatite series. A CaHPO(4) treatment combined with apatite or MBM significantly decreased solubility of P from that of CaHPO(4) singly. Tests with six additional genotypes showed that all solubilised P from CaHPO(4), some to a significant extent from apatite, MBM or slightly from FePO(4), but none from AlPO(4). The accumulation of nearly water-insoluble aluminium and iron phosphates in field and virgin soils is partly explainable by the immobilisation through the root action on P, which we have found also with other plant species. The root responses must also have ecophysiological functions distinct from P acquisition.

New advances in nanocrystalline apatite colloids intended for cellular drug delivery.

Intracellular drug delivery using colloidal biomimetic calcium phosphate apatites as nanocarriers is a seducing concept. However, the colloid preparation to an industrial scale requires the use of easily handled raw materials as well as the possibility to tailor the nanoparticles size. In this work, the stabilization of the colloids was investigated with various biocompatible agents. Most interestingly, nanoscale colloids were obtained without the need for toxic and/or hazardous raw materials. Physico-chemical characteristics were investigated by chemical analyses, dynamic light scattering, FTIR/Raman spectroscopies, XRD, and electron microscopy. A particularly promising colloidal system associates biomimetic apatite stabilized with a natural phospholipid moiety (AEP(r), 2-aminoethylphosphoric acid). Complementary data described such colloids as apatite nanocrystals covered with surface Ca(2+)(AEP(r)(-))(2) complexes involving "supernumerary" Ca(2+) ions. The effects of the concentration in AEPr, synthesis temperature, duration of aging in solution, pH, and sonication were followed, showing that it is possible to modulate the mean size of the nanoparticles, typically in the range 30-100 nm. The perfect biocompatibility of such colloids allied to the possibility to prepare them from innocuous compounds shows great promise for intracellular drug delivery.

Localisation of phosphomonoesterase activity in ectomycorrhizal fungi grown on different phosphorus sources.

Phosphorus (P) is a major limiting nutrient for plants in boreal forest ecosystems where a substantial part of the total P is sequestered in organic compounds. Some ectomycorrhizal (ECM) fungi are known to produce phosphomonoesterases, enzymes that degrade organic P sources. Here, we test 16 ECM species for this enzymatic activity by growing them on media containing orthophosphate, phytic acid or apatite. A method with an overlay gel that determined both phosphomonoesterase activity and its spatial distribution was developed. The phosphomonoesterase activity was not significantly higher when growing on organic P; conversely some isolates only produced measurable enzyme activity when grown on apatite. Species-specific variations with respect to phosphomonoesterase activity as well as growth responses to different substrates were found. The production of phosphomonoesterases was found to be widespread in ECM fungi and the enzyme activity did not need induction by organic P. The enzyme activity was highest in the central parts of the mycelia, potentially reflecting breakdown and recycling of phospholipids from old hyphae or potentially higher mycelial density.

Ectomycorrhizal mycelial species composition in apatite amended and non-amended mesh bags buried in a phosphorus-poor spruce forest.

We studied the effect of apatite amendment on ectomycorrhizal (EM) mycelial biomass production and species composition in a phosphorus-poor spruce forest using sand-filled mesh bags. Control and apatite-amended bags were buried in pairs in the lower part of the organic horizon for one growth season. DNA extraction, PCR of the ITS region, cloning and random sequencing were used to examine the fungal species composition in each bag. Sequences were identified by comparison with the UNITE database and GenBank. Our study confirmed previous results that the major fungal ingrowth in mesh bags was of EM origin. On average 13 species were detected in each bag. Tylospora fibrillosa, Amphinema sp., Tomentellopsis submollis, and Xerocomus badius made up almost 80% of the EM sequences. High biomass was related to increased dominance of specific species. There were no statistically significant differences in biomass production estimated from PLFA 18:2omega6, 9, or between fungal communities of apatite-amended and control bags estimated from DNA after one growth season. The potential of the mesh bag method in studies of functional diversity of EM mycelia in the field is discussed.

[Experimental study of the mechanism for vascular calcification in chronic kidney disease]. / Experimenteel onderzoek naar de mechanismen van vaatverkalking bij chronisch nierlijden.

Vascular calcification or ectopic calcification ofblood vessels forms an important element of the increased cardiovascular risk observed in patients with chronic kidney disease. In addition to the classical Framingham risk factors, specific uremia-related factors such as hyperphosphatemia and disturbed calcium and phosphorus metabolism contribute to the development of vascular calcification. To gain a better insight into the mechanism of this calcification process, experimental techniques were developed to induce and detect vascular calcification in rats with in vivo micro-CT imaging. By means of synchrotron-based micro-X-ray diffraction the mineral phase deposited in arteries of rats with adenine-induced chronic renal failure was found to consist mainly of hydroxyapatite, whereas calcifications induced with high dose vitamin D administration additionally contained whitlockite, a magnesium-containing mineral. Vascular calcification is an active, cell-regulated process. By immunohistochemically investigating the expression of bone-specific proteins in calciying arteries, we demonstrated that calcifying vascular smooth muscle cells are not only able to acquire an osteoblast-like phenotype, but can moreover transdifferentiate to chondrocyte-like cells, expressing the cartilage transcription factor sox9 and the cartilage extracellular matrix protein collagen II. This cartilage phenotype was also found in human aortic tissue. Finally, treatment of uremic rats with the calcium-free phosphate binder lanthanum carbonate was shown to inhibit the development of vascular calcification, implying that adequate phosphorus control without additional calcium load reduces vascular calcification. In the future, we will map the proteome of calcifying vascular smooth muscle cells and investigate the paradoxical association of vascular calcification with impaired bone mineralisation.

Estimation of the specific surface area of apatites in human mineralized tissues using 31P MAS NMR.

Specific surface areas of apatites in whole human mineralized tissues were estimated from (31)P MAS NMR linewidths: 77 m(2)g(-1) for enamel and 94 m(2)g(-1) for dentin, dental cementum and cortical bone.

Trophic level and macronutrient shift effects associated with the weaning process in the Postclassic Maya.

The weaning process was investigated at two Maya sites dominated by Postclassic remains: Marco Gonzalez (100 BC-AD 1350) and San Pedro (1400-AD 1650), Belize. Bone collagen and bioapatite were analyzed from 67 individuals (n < or = 6 years = 15, n > 6 years = 52). Five isotopic measures were used to reconstruct diet and weaning: stable nitrogen- and carbon-isotope ratios in collagen, stable carbon- and oxygen-isotope ratios in bioapatite, and the difference in stable carbon-isotope values of coexisting collagen and bioapatite. Nitrogen-isotope ratios in infant collagen from both sites are distinct from adult females, indicating a trophic level effect. Collagen-to-bioapatite differences in infant bone from both sites are distinct from adult females, indicating a shift in macronutrients. Oxygen-isotope ratios in infant bioapatite from both sites are also distinct from adult females, indicating the consumption of breast milk. Among infants, carbon- and nitrogen-isotope ratios vary, indicating death during different stages in the weaning process. The ethnohistoric and paleopathological literature on the Maya indicate cessation of breast-feeding between ages 3-4 years. Isotopic data from Marco Gonzalez and San Pedro also indicate an average weaning age of 3-4 years. Based on various isotopic indicators, weaning likely began around age 12 months. This data set is not only important for understanding the weaning process during the Postclassic, but also demonstrates the use of collagen-to-bioapatite spacing as an indicator of macronutrient shifts associated with weaning.

Ectomycorrhizal fungal biomass in roots and uptake of P from apatite by Pinus sylvestris seedlings growing in forest soil with and without wood ash amendment.

Forest soil from an experimental Norway spruce forest with four levels of wood ash addition (0, 1, 3 and 6 tonnes ha(-1)) was used to inoculate pine (Pinus sylvestris) seedlings with indigenous ectomycorrhizal (EM) fungi. Uptake of 32P and 86Rb in a root bioassay was used to estimate the demand for P and K by seedlings grown in the different soils. Utilisation of P from apatite was tested in a laboratory system where uptake by the ectomycorrhizal mycelium was separated from uptake by roots. The demand for P and K in the seedlings was similar regardless of the ash treatment. Variation in EM levels, estimated as fungal biomass (ergosterol) in roots, was large in the different soils, but not related to ash addition. Uptake of P from apatite was, on average, 23% of total seedling P and was not related to EM levels. It was concluded that the improved P uptake from apatite by EM fungi found in earlier studies is probably not a general phenomenon among EM fungi. The small effect of ash addition on EM levels and P uptake suggests that addition of granulated wood ash is a forest management treatment that will have only minor influence on ectomycorrhizal symbiosis.

Biological factors in dental caries: role of remineralization and fluoride in the dynamic process of demineralization and remineralization (part 3).

Dental caries is a complex disease process that afflicts a large proportion of the world, regardless of gender, age and ethnicity, although it does tend to affect more with a low socioeconomic status to a greater extent. Remineralization may be enhanced by providing low levels of calcium and phosphate, in conjunction with minimal amounts of fluoride. It is truly remarkable the difference that a very small amount of fluoride (<1 ppm) has upon demineralization and remineralization. This is because fluoride acts as a catalyst and influences reaction rates with dissolution and transformation of various calcium phosphate mineral phases within tooth structure and resident within plaque adjacent to tooth surfaces. The incorporation of minimal amounts of fluoride into HAP yields FHAP that resists demineralization to similar level as FAP. New and emerging methods have been and are in the process of being developed. These hold great promise for preventing and reversing caries, especially in the one-fifth of the population that accounts for two-thirds of the caries experience. Still, the mainstay in caries prevention and remineralization is frequent exposure to low levels of fluoride. This may be accomplished with fluoridated toothpastes, supplemented with fluoride mouthrinses, CPP-ACP containing chewing gum and application of fluoride varnishes. The role of systemic fluorides appears to be limited and primarily has a topical effect.

Rapidly forming apatitic mineral in an osteoblastic cell line (UMR 106-01 BSP).

This study evaluated a rapid biomineralization phenomenon exhibited by an osteoblastic cell line, UMR 106-01 BSP, when treated with either organic phosphates [beta-glycerophosphate (beta-GP), Ser-P, or Thr-P], inorganic phosphate (P(i)), or calcium. In a dose-dependent manner, these agents (2-10 mM) stimulated confluent cultures to deposit mineral in the cell layer (ED50 of approximately 4.6 mM for beta-GP (30 +/- 2 nmol Ca2+/microgram DNA) and approximately 3.8 mM (29 +/- 2 nmol Ca2+/microgram DNA) for P(i)) with a plateau in mineral formation by 20 h (ET50 approximately 12-15 h). beta-GP or P(i) treatment yielded mineral crystals having an x-ray diffraction pattern similar to normal human bone. Alizarin red-S histology demonstrated calcium mineral deposition in the extracellular matrix and what appeared to be intracellular paranuclear staining. Electron microscopy revealed small, needle-like crystals associated with fibrillar, extracellular matrix deposits and intracellular spherical structures. Mineral formation was inhibited by levamisole (ED50 approximately 250 microM), pyrophosphate (ED50 approximately 1-10 microM), actinomycin C1 (500 ng/ml), cycloheximide (50 micrograms/ml), or brefeldin A (1 microgram/ml). These results indicate that UMR 106-01 BSP cells form a bio-apatitic mineralized matrix upon addition of supplemental phosphate. This process involves alkaline phosphatase activity, ongoing RNA and protein synthesis, as well as Golgi-mediated processing and secretion.