45Ca Incorporation Research Articles

Intercomparison of four methods to estimate coral calcification under various environmental conditions

Abstract. Coral reefs are constructed by calcifiers that precipitate calcium carbonate to build their shells or skeletons through the process of calcification. Accurately assessing coral calcification rates is crucial to determine the health of these ecosystems and their response to major environmental changes such as ocean warming and acidification. Several approaches have been used to assess rates of coral calcification, but there is a real need to compare these approaches in order to ascertain that high-quality and intercomparable results can be produced. Here, we assessed four methods (total alkalinity anomaly, calcium anomaly, 45Ca incorporation, and 13C incorporation) to determine coral calcification of the reef-building coral Stylophora pistillata. Given the importance of environmental conditions for this process, the study was performed under two starting pH levels (ambient: 8.05 and low: 7.2) and two light (light and dark) conditions. Under all conditions, calcification rates estimated using the alkalinity and calcium anomaly techniques as well as 45Ca incorporation were highly correlated. Such a strong correlation between the alkalinity anomaly and 45Ca incorporation techniques has not been observed in previous studies and most probably results from improvements described in the present paper. The only method which provided calcification rates significantly different from the other three techniques was 13C incorporation. Calcification rates based on this method were consistently higher than those measured using the other techniques. Although reasons for these discrepancies remain unclear, the use of this technique for assessing calcification rates in corals is not recommended without further investigations.

Cell Biological Assays for Measuring Chondrogenic Activities of CCN2 Protein.

Growth-plate chondrocytes undergo proliferation, maturation, hypertrophic differentiation, and calcification; and these processes can be reproduced in vitro in a chondrocyte culture system. Using this system, we have shown that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes all stages of proliferation, maturation, hypertrophic differentiation, and calcification, thus suggesting that CCN2 is a multifunctional growth factor for chondrocytes and plays important roles in chondrocyte proliferation and differentiation. In this chapter, we describe how to evaluate CCN2 functions in these processes occurring in cultured chondrocytes. Evaluation strategies for cell proliferation include measuring DNA synthesis by [3H]-thymidine incorporation, cellular metabolic activity, and cell number with a hemocytometer. Next, evaluation strategies to assess maturation are analysis of the gene expression of markers of mature chondrocytes, and examination of proteoglycan and collagen synthesis by using radioactive compounds. In addition, cytohistochemical detection of glycosaminoglycans (GAGs), such as chondroitin sulfate, by use of alcian blue and toluidine blue staining is useful to evaluate chondrocyte maturation. These methods can be also used for evaluation of physiological functions of CCN2 in permanent chondrocytes such as articular and auricular chondrocytes, which do not calcify under physiological conditions. Next, evaluation of hypertrophic differentiation is performed by detecting type X collagen, which is specific marker of hypertrophic chondrocytes, and by measuring alkaline phosphatase (ALP) activity. Finally, evaluation of calcification is performed by detecting matrix calcification by use of alizarin red staining and by examining the incorporation of 45Ca into cartilaginous matrix. These methods would be useful for the evaluation not only of CCN2 but also of its derivatives and other CCN proteins.

Ocean acidification reshapes the otolith-body allometry of growth in juvenile sea bream

The effects of elevated CO2 partial pressure (pCO2) on otolith calcification and on the coupling between somatic and otolith growth were investigated in juvenile gilthead sea bream Sparus aurata. Six-month old individuals were raised during seven weeks under four pCO2 conditions set according to projected future ocean acidification scenarios. Body and otolith biometric parameters were measured throughout the experiment along with the otolith biomineralization monitored using a radiotracer technique based on 45Ca incorporation. Sea bream exhibited somatic growth resilience to all treatments. In contrast, increased growth rate and shape complexity of otoliths were observed with a pHT drop from 8.1 to 7.5. Hypercalcification was observed under lowered pH, with a rate of calcium incorporation increasing by up to 18% between pHT 8.1 and pHT 7.7. This work highlighted an uncoupling of otolith and body growth of juvenile sea bream within 40days at pHT 7.9 projected to be reached by the end of the century. As the otolith is an essential tool used in reconstructing fish life history, this work suggests that information resulting from otolith studies should be interpreted with caution with respect to the potential impacts that ocean acidification projected modifications could have on otolith biomineralization.

Sublethal mechanisms of Pb and Zn toxicity to the purple sea urchin (Strongylocentrotus purpuratus) during early development

In order to understand sublethal mechanisms of lead (Pb) and zinc (Zn) toxicity, developing sea urchins were exposed continuously from 3h post-fertilization (eggs) to 96h (pluteus larvae) to 55 (±2.4)μgPb/L or 117 (±11)μgZn/L, representing ∼70% of the EC50 for normal 72h development. Growth, unidirectional Ca uptake rates, whole body ion concentrations (Na, K, Ca, Mg), Ca2+ ATPase activity, and metal bioaccumulation were monitored every 12h over this period. Pb exhibited marked bioaccumulation whereas Zn was well-regulated, and both metals had little effect on growth, measured as larval dry weight, or on Na, K, or Mg concentrations. Unidirectional Ca uptake rates (measured by 45Ca incorporation) were severely inhibited by both metals, resulting in lower levels of whole body Ca accumulation. The greatest disruption occurred at gastrulation. Ca2+ ATPase activity was also significantly inhibited by Zn but not by Pb. Interestingly, embryos exposed to Pb showed some capacity for recovery, as Ca2+ATPase activities increased, Ca uptake rates returned to normal intermittently, and whole body Ca levels were restored to control values by 72–96h of development. This did not occur with Zn exposure. Both Pb and Zn rendered their toxic effects through disruption of Ca homeostasis, though likely through different proximate mechanisms. We recommend studying the toxicity of these contaminants periodically throughout development as an effective way to detect sublethal effects, which may not be displayed at the traditional toxicity test endpoint of 72h.

Genetically Modified Medicago truncatula Lacking Calcium Oxalate has Increased Calcium Bioavailability and Partially Rescues Vitamin D Receptor Knockout Mice Phenotypes

Background: How the distribution and sequestered form of plant macro/micro-nutrients influence their bioavailability, and ultimately impact human health, are poorly understood. The legume Medicago truncatula has a portion of its tissue calcium (Ca) sequestered in the form of the Ca oxalate (CaOx) crystal which reduces its nutritional value in terms of Ca bioavailability. The calcium oxalate deficient 5 (cod5) mutant has a total Ca content similar to wild-type (WT) plants, but sequesters less of its tissue Ca in the form of the CaOx crystal. Previous short-term mice feeding studies suggest that this difference is responsible for the improved Ca bioavailability of the cod5 plants compared to WT plants. Objectives: To perform long term feeding studies with Vitamin D Receptor Knockout (VDR-KO) mice and the nutritionally improved cod5 line to assess the impact of increased Ca bioavailability on VDR-KO Ca deficiency phenotypes. Methods: To assess the ability of diets containing cod5 plant material as the sole Ca source to rescue the Ca deficiency phenotypes of the VDR-KO mice we conducted both short term and long term experiments. Specifically, Ca absorption and utilization were measured short term (24-hour) in the hind limb bones and duodenum tissue of VDR-KO mice that were fed either an intrinsically 45Ca labeled cod5 or WT Medicago diet. Long term (20-day) bodyweight gain and change in Bone Mineral Density (BMD) were also measured over a 20 day period in VDR-KO mice fed either a cod5 or WT Medicago diet. Results: In the 24-hour feeding study, 45Ca incorporation was found to be 46.3% (male) or 53.9% (female) higher in hind limb bones (P<0.01); and 32.5% (male) or 38.5% (female) higher in duodenums (P<0.01) in VDR-KO mice fed cod5 than those fed WT plants. In the 20-day feeding study, the VDR-KO mice (male) fed cod5 gained 38.1% more bodyweight than those fed WT plants (P=0.06). The increase of BMD after 20 days in the VDR-KO mice (male) fed cod5 diets was 22.5% higher than those fed WT diets (P=0.17). Conclusions: Our study confirms and extends an earlier study by showing that cod5 Medicago not only had higher Ca bioavailability but it can also rescue, in part, the VDR-KO Ca deficiency phenotypes. Thus, the removal of CaOx from a plant-based diet appears to be a viable long-term dietary option to boost bioavailable Ca levels and help combat Ca related disorders.

Ocean acidification and temperature rise: effects on calcification during early development of the cuttlefish Sepia officinalis

This study investigated the effects of seawater pH (i.e., 8.10, 7.85 and 7.60) and temperature (16 and 19 °C) on (a) the abiotic conditions in the fluid surrounding the embryo (viz. the perivitelline fluid), (b) growth, development and (c) cuttlebone calcification of embryonic and juvenile stages of the cephalopod Sepia officinalis. Egg swelling increased in response to acidification or warming, leading to an increase in egg surface while the interactive effects suggested a limited plasticity of the swelling modulation. Embryos experienced elevated pCO2 conditions in the perivitelline fluid (>3-fold higher pCO2 than that of ambient seawater), rendering the medium under-saturated even under ambient conditions. The growth of both embryos and juveniles was unaffected by pH, whereas 45Ca incorporation in cuttlebone increased significantly with decreasing pH at both temperatures. This phenomenon of hypercalcification is limited to only a number of animals but does not guarantee functional performance and calls for better mechanistic understanding of calcification processes.

Effects of increased pCO2 on zinc uptake and calcification in the tropical coral Stylophora pistillata

Zinc (Zn) is an essential element for corals. We investigated the effects of ocean acidification on zinc incorporation, photosynthesis, and gross calcification in the scleractinian coral Stylophora pistillata. Colonies were maintained at normal pHT (8.1) and at two low-pH conditions (7.8 and 7.5) for 5 weeks. Corals were exposed to 65Zn dissolved in seawater to assess uptake rates. After 5 weeks, corals raised at pHT (8.1) exhibited higher 65Zn activity in the coral tissue and skeleton, compared with corals raised at a lower pH. Photosynthesis, photosynthetic efficiency, and gross calcification, measured by 45Ca incorporation, were however unchanged even at the lowest pH.

Lyso-phosphatidylcholine induces osteogenic gene expression and phenotype in vascular smooth muscle cells

ObjectiveCalcifying vascular cells in human atherosclerotic plaques actively contribute to ectopic vascular mineralization. Lyso-phosphatidylcholine (LPC), a product of oxidized phosphatidylcholine hydrolysis, is found at concentrations of 1–12μg/g tissue throughout the atheroma. The objective of this study was to determine if LPC induces an osteogenic phenotype in vascular smooth muscle cells. Methods and resultsProliferating human aortic smooth muscle cells were treated with a wide-range of LPC concentrations (0.1nM to 100μM) over 14 days. Von Kossa, Alizarin Red S, and alkaline phosphatase staining were used to identify mineralizations. RT-PCR, ELISA, alkaline phosphatase activity, and 45Ca incorporation assays were used to evaluate the osteo-inductive effect of LPC on smooth muscle phenotype. Histology and morphometry revealed that cells treated with as little as 10nM LPC produced calcium phosphate deposits in culture. LPC-treated vascular smooth muscle cells showed a significant increase in 45Ca incorporation and alkaline phosphatase activity. Furthermore, LPC treatment induced a significant loss of Schnurri 3 protein, a key repressor of Runt-related transcription factor 2 stability. Genomic studies revealed that osteogenic gene expression was significantly up-regulated in LPC-treated cells, which is attributed to increased Runt-related transcription factor 2 expression and transcriptional activity. ConclusionLPC induces osteogenic morphology, physiology, gene expression, and phenotype in vascular smooth muscle cells. The present study suggests that localized concentrations of LPC in human atherosclerotic plaques may be a contributing factor to the generation of calcifying vascular cells.

Calcification of the cold-water coral &amp;lt;i&amp;gt;Lophelia pertusa,&amp;lt;/i&amp;gt; under ambient and reduced pH

Abstract. The cold-water coral Lophelia pertusa is one of the few species able to build reef-like structures and a 3-dimensional coral framework in the deep oceans. Furthermore, deep cold-water coral bioherms may be among the first marine ecosystems to be affected by ocean acidification. Colonies of L. pertusa were collected during a cruise in 2006 to cold-water coral bioherms of the Mingulay reef complex (Hebrides, North Atlantic). Shortly after sample collection onboard these corals were labelled with calcium-45. The same experimental approach was used to assess calcification rates and how those changed due to reduced pH during a cruise to the Skagerrak (North Sea) in 2007. The highest calcification rates were found in youngest polyps with up to 1% d−1 new skeletal growth and average rates of 0.11±0.02% d−1±S.E.). Lowering pH by 0.15 and 0.3 units relative to the ambient level resulted in calcification being reduced by 30 and 56%. Lower pH reduced calcification more in fast growing, young polyps (59% reduction) than in older polyps (40% reduction). Thus skeletal growth of young and fast calcifying corallites suffered more from ocean acidification. Nevertheless, L. pertusa exhibited positive net calcification (as measured by 45Ca incorporation) even at an aragonite saturation state (Ωa) below 1.

Further findings on the inhibitory effect of estradiol benzoate on the circulation and on 45Ca and 3H-proline incorporation in rat bones.

Various aspects of the effect of estradiol benzoate (EB, Agofollin Depot, Czechoslovakia, usually in a s.c. dose of 5 mg/kg body weight once a week), on the local circulation (the uptake of 85Sr-microspheres), the incorporation of 45Ca and 3H-proline, bone density and ash weight related to bone volume were studied in six experiments in the tibia and distal femur of 224 rats. 1. The dose-response. In rats treated four weeks with doses of 0.5, 1.0, 1.5 and 2.0 mg EB per rat once a week, a significant correlation with the uptake of 85Sr-microspheres (r = -0.56), the blood flow (r = -0.56), the incorporations of 45Ca (r = -0.89) and 3H-proline (r = -0.35) and body weight (r = -0.71) was demonstrated. 2. The time course of changes after 1, 2, 4 and 8 weeks administration of EB. Circulatory values were not significantly lowered until after four weeks, 45Ca incorporation and body weight were significantly lower after only one week and 3H-proline incorporation did not fall until after eight weeks. The course of the uptake of 85Sr-microspheres and the incorporation of 45Ca were very similar. 3. Comparison of males and females. EB reduced circulatory values and 45Ca and 3H-proline incorporation and increased bone density and bone ash weight in both males and females. Conclusions. The decrease in the local bone blood flow after EB showed a significant dose-effect correlation; the decrease in circulatory values, on using the given administration method, is significant after four weeks. The inhibitory effect of EB on the bone blood flow is not sex-specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible participation of prostaglandins in the increase in the bone blood flow in oophorectomized female rats.

The aim of this study was to verify the possible role of prostaglandin (PG) in the increase in the bone blood flow of female rats after oophorectomy (OOX). In two experiments we determined blood flow in the tibia and distal femur (85Sr-microspheres) and 24-h incorporation of 45Ca and 3H-proline. Acetylosalicyclic acid (ASA, 0.13% in the food for 4 weeks) was used to suppress the production of PG. There was an increase in the bone blood flow after OOX (performed 4 weeks prior to the experiment), no change after ASA alone and significant reduction by ASA of the OOX-induced increase in the bone blood flow. In both groups of OOX females there was a decrease in tibial bone density and ash weight. The changes in 45Ca incorporation were similar to those in the blood flow while the changes in 3H-proline incorporation were not significant. Thus, the effect of ASA, i.e. suppression of the OOX-induced increase in the bone blood flow, is consistent with the possible role of PG (probably PGE2) in the increase in bone blood flow of OOX female rats.

Spraying calcium is not an effective way to increase structural calcium in litchi pericarp

Calcium in different forms was compared in the pericarp of cracked and healthy fruit in the cracking-susceptible litchi ( Litchi chinensis Sonn.) cultivar ‘Nuomici’. Incorporation of 45Ca into the cell walls of the pericarp and effects of sprays of calcium chloride (0.2% or 0.5%) on endogenous calcium and cracking incidence were examined. The results showed that calcium concentration in cracked fruit was significantly lower than in healthy fruit. Structural calcium as part of cell wall components contributed to the majority of the difference in calcium concentration between cracked fruit and healthy fruit. Surface spray of calcium chloride, either once or three times at different stages of fruit development exhibited no significant effect on fruit cracking and hardly increased calcium, especially structural calcium in the pericarp. X-ray microanalysis revealed that the applied calcium remained on pericarp surface. Radioisotope test showed that less than 0.1% of 45Ca applied on fruit surface was incorporated into the cell walls. It is suggested that spraying calcium on fruit surface is not an effective way to prevent litchi fruit cracking due to the difficulty in uptake of the calcium sprayed on fruit surface.

Upper thermal thresholds of shallow vs. deep populations of the precious Mediterranean red coral Corallium rubrum (L.): Assessing the potential effects of warming in the NW Mediterranean

Recent mortality outbreaks in marine ecosystems have been linked to elevated seawater temperatures associated with global climate change. Acquisition of thermotolerance data is essential, not only to determine the role of temperature in mortality outbreaks, but also to predict consequences of global warming. In the NW Mediterranean region, elevated seawater temperatures during the summer periods of 1999 and 2003 caused mass mortality of the Mediterranean red coral, Corallium rubum (L. 1758). Experiments testing the upper thermal limits of this species were carried out in aquaria using samples collected from populations from 11 to 40 m depth in the Marseilles region (NW Mediterranean, France). Samples were subjected to temperature treatments between 18 and 30 °C with an exposure time of 5 and 25 days. Three biological response variables were used to evaluate effects of the treatments: coenenchyme necrosis, polyp activity and calcification rates ( 45Ca incorporation in calcareous skeleton). The results showed that exposure to 24 °C for 24 days caused a beginning of mortality only for the deep population, and to 25 °C for between 9 and 14 days caused mass mortality of both sample groups. The response variable results indicate that samples from the shallow population had greater thermotolerance of elevated seawater temperatures than the deep samples. The shallow samples showed greater polyp activity and higher calcification rate with a delayed necrosis response than the deep samples. These initial thermotolerance results combined with both hydrographic models and seawater temperature monitoring are the first step towards developing predictive tools for anticipating future effects of climate change in the red coral populations.

Extracellular matrix production and calcium carbonate precipitation by coral cells in vitro

The evolution of multicellularity in animals required the production of extracellular matrices that serve to spatially organize cells according to function. In corals, three matrices are involved in spatial organization: (i) an organic ECM, which facilitates cell-cell and cell-substrate adhesion; (ii) a skeletal organic matrix (SOM), which facilitates controlled deposition of a calcium carbonate skeleton; and (iii) the calcium carbonate skeleton itself, which provides the structural support for the 3D organization of coral colonies. In this report, we examine the production of these three matrices by using an in vitro culturing system for coral cells. In this system, which significantly facilitates studies of coral cell physiology, we demonstrate in vitro excretion of ECM by primary (nondividing) tissue cultures of both soft (Xenia elongata) and hard (Montipora digitata) corals. There are structural differences between the ECM produced by X. elongata cell cultures and that of M. digitata, and ascorbic acid, a critical cofactor for proline hydroxylation, significantly increased the production of collagen in the ECM of the latter species. We further demonstrate in vitro production of SOM and extracellular mineralized particles in cell cultures of M. digitata. Inductively coupled plasma mass spectrometry analysis of Sr/Ca ratios revealed the particles to be aragonite. De novo calcification was confirmed by following the incorporation of (45)Ca into acid labile macromolecules. Our results demonstrate the ability of isolated, differentiated coral cells to undergo fundamental processes required for multicellular organization.

Chemical and hormonal determinants of vascular calcification in vitro

Vascular calcification is a complex process that is dependent not only on the physicochemical effects of Ca, PO(4), and pH, but also on smooth muscle factors that may be regulated by these ions as well as by 1,25-dihydroxyvitamin D(3) (calcitriol) and parathyroid hormone (PTH). These minerals and hormones were tested in a model of medial calcification in rat aorta maintained in culture for 9 days. Calcification was quantitated as incorporation of (45)Ca, alkaline phosphatase activity was measured in aortic homogenates, and osteopontin production was measured from immunoblots of culture medium. At 1.8 mM Ca (1.46 mM free), calcification occurred at or above 2.8 mM PO(4). At 3.8 mM PO(4), calcification occurred at or above 1.10 mM free [Ca]. At a constant [Ca] x [PO(4)], calcification varied directly with [Ca] and inversely with [PO(4)]. Calcification was directly related to pH between 7.19 and 7.50 but not altered by PTH or calcitriol. Alkaline phosphatase activity and osteopontin production were increased by Ca, PO(4), calcitriol, and PTH. We conclude that calcification of rat aorta in vitro requires elevation of both [Ca] and [PO(4)], and that [Ca] rather than [PO(4)] or the product of the two is the dominant determinant. The induction of alkaline phosphatase and osteopontin indicates that Ca and PO(4) have effects in addition to simple physicochemical actions. Although PTH and calcitriol did not increase calcification in vivo, they have effects on smooth muscle that could influence calcification in vivo. Calcification is enhanced by alkalinity within the range produced during hemodialysis.

Human endothelial cells inhibit BMSC differentiation into mature osteoblasts in vitro by interfering with osterix expression

It is well accepted, that there is communication between osteoblasts and endothelial cells. However, the influence of endothelial cells on the differentiation of bone precursors into mature osteoblasts is not yet well understood. We therefore studied the effect of human umbilical vein endothelial cells (HUVEC) on human bone marrow stromal cell (BMSC) differentiation towards an osteoblastic phenotype by culturing them in two different types of HUVEC-BMSC cultures (indirect contact, HUVEC-conditioned medium). Typical bone markers (45Ca incorporation, ALP activity, and gene expression of collagen 1 (COL1), osteonectin (ON), matrix metalloproteinase 13 (MMP-13), bone morphogenetic protein 2 (BMP-2)) and transcription factors (Runx2, osterix (OSX)) were evaluated at different time points. The effect of stimulating HUVEC with vascular endothelial growth factor (VEGF) before co-cultures with BMSC was also evaluated. As expected, BMSC in osteogenic medium (OM) (with dexamethasone) differentiated towards the osteoblastic phenotype, as measured by increased matrix mineralization, high ALP activity, and elevated expression of specific osteoblastic marker genes, when compared to BMSC in non-OM. HUVEC reversibly inhibited osteoblastic differentiation of BMSC in OM, independent of the co-culture type. When HUVEC were removed from the cultures, BMSC differentiated into osteoblasts, albeit at a faster rate than BMSC that were never co-cultured with HUVEC. Stimulation of HUVEC with VEGF before co-culture enhanced the inhibitory effect of HUVEC on BMSC differentiation. This inhibitory effect was connected to a reversible suppression of OSX gene expression and was specific to endothelial cells. We conclude that HUVEC can inhibit dexamethasone-induced BMSC differentiation into osteoblasts in vitro, by interfering with OSX expression, thereby arresting BMSC differentiation at a preosteoblastic stage.

Indomethacin inhibits bone formation in inductive allografts but not in autografts

Background Non-steroidal anti-inflammatory drugs (NSAIDs) are known to have inhibitory effects on new bone formation. We wanted to analyze some effects of the NSAID indomethacin on different inductive stimuli for bone formation.Methods We experimentally induced heterotopic new bone with demineralized allogeneic bone matrix (DABM) and with bone autografts in rats, in order to study the effects of indomethacin on new bone formation at 3 and 6 weeks.Results Indomethacin inhibited net bone formation in DABMs by 30% at 6 weeks. At 6 weeks, the mineral accretion rate was unaffected, indicating that it is at the early phase of the inductive process that mineral accretion is sensitive to inhibition by indomethacin, but not at the later stages.In traumatized skeletal bone, the 45Ca-specific activity was higher than in non-traumatized bone, while indomethacin significantly reduced the 45Ca uptake at 3 weeks, but not at 6 weeks.In the autografts, a net mineral loss occurred, but neither mineral content nor 45Ca incorporation was affected by Indomethacin treatment.Interpretation Indomethacin inhibited the early phase of new bone formation in heterotopic DABMs and the early bone healing process in traumatized skeletal bone, but did not affect resorption or bone formation in heterotopic autografts. ▪

Calbindin Independence of Calcium Transport in Developing Teeth Contradicts the Calcium Ferry Dogma

Cytosolic calcium-binding proteins termed calbindins are widely regarded as a key component of the machinery used to transport calcium safely across cells. Acting as mobile buffers, calbindins are thought to ferry calcium in bulk and simultaneously protect against its potentially cytotoxic effects. Here, we contradict this dogma by showing that teeth and bones were produced normally in null mutant mice lacking calbindin(28kDa). Structural analysis of dental enamel, the development of which depends critically on active calcium transport, showed that mineralization was unaffected in calbindin(28kDa)-null mutants. An unchanged rate of calcium transport was verified by measurements of (45)Ca incorporation into developing teeth in vivo. In enamel-forming cells, the absence of calbindin(28kDa) was not compensated by other cytosolic calcium-binding proteins as detectable by (45)Ca overlay, two-dimensional gel, and equilibrium binding analyses. Despite a 33% decrease in cytosolic buffer capacity, cytotoxicity was not evident in either the null mutant enamel or its formative cells. This is the first definitive evidence that calbindins are not required for active calcium transport, either as ferries or as facilitative buffers. Moreover, in challenging the broader notion of a cytosolic route for calcium, the findings support an alternative paradigm involving passage via calcium-tolerant organelles.

The effect of different mineral frames on ectopic bone formation in mouse hind leg muscles induced by native reindeer bone morphogenetic protein

Bone morphogenetic proteins (BMPs) require carrier material for slow release and framing material for osteoconduction. The effect of a frame on early bone formation induced by partially purified native reindeer BMP in composite implants containing 3 mg of BMP, type IV collagen and tricalcium phosphate (TCP/Col/BMP) or hydroxyapatite (HA/Col/BMP) or biphasic tricalcium phosphate-hydroxyapatite (TCP/HA/Col/BMP) or biocoral (NC/Col/BMP) was evaluated using a mouse hind leg muscle pouch model. Collagen with native reindeer BMP (Col/BMP) and corresponding implants without native reindeer BMP served as controls. Evaluation was done by incorporation of 45Ca, radiographically and histologically 3 weeks after the implantation. None of the implants without native reindeer BMP were able to induce new bone visible on radiographs. The area of new bone formation in the Col/BMP (p=0.026) and TCP/HA/Col/BMP (p=0.012) groups was significantly greater than in the TCP/Col/BMP group. The optical density of the new bone area was significantly greater in the TCP/HA/Col/BMP group than in the TCP/Col/BMP (p=0.036) or Col/BMP (p=0.02) groups. 45Ca incorporation was many times greater in all the groups containing native reindeer BMP than in the corresponding groups without BMP. In the Col/BMP (p=0.046) and TCP/HA/Col/BMP (p=0.046) groups, 45Ca incorporation was significantly greater than in the TCP/Col/BMP group. No significant differences were found in any parameters between HA/Col/BMP and NC/Col/BMP groups and the other BMP-containing groups. Hydroxyapatite, biocoral and biphasic tricalciumphosphate-hydroxyapatite are equally good as framing material for native reindeer BMP, while tricalciumphosphate is somewhat worse. Osteoinduction of native reindeer BMP works well with collagen alone.

Effects of PHEX antisense in human osteoblast cells.

X-linked hypophosphatemia (XLH) is an X-linked dominant disorder that is characterized by rachitic bone disease and hypophosphatemia due to renal phosphate transport defect. The candidate gene for XLH, PHEX, has recently been identified and found to share high homology with endopeptidases. PHEX is expressed in various tissues, including bones, and the available evidence today indicates that bones can release abnormal humoral factors that affect bone mineralization and proximal tubule phosphate transport in XLH. It was, therefore, hypothesized that the inactivating mutations of PHEX in bone may lead to the release of humoral factors and contribute to the phenotypic expression of the disease. To test this possibility, clones of MG-63 cells, a human osteoblast cell line, were produced and stably transfected with PHEX-antisense vectors, resulting in a decrease in PHEX expression at mRNA and protein levels. It was found that these antisense-transfected cells had impaired mineralization, with a decrease in 45Ca incorporation and calcification nodule formation. It was also found that the conditioned culture media collected from these antisense-transfected cells exhibited inhibitory activities on 45Ca incorporation by the nontransfected MG-63 cells and 32P uptake by the opossum kidney proximal tubular cells. The results of the study, therefore, provide strong evidence that supports the link between PHEX mutations and the pathogenesis of XLH.