Mineral Crystal Growth Research Articles

Evaluation of Natural Organic Additives as Eco-friendly Inhibitors for Calcium and Magnesium Scale Formation in Water Systems.

Mineral scale formation reduces the heat transfer efficiency and clogs pipes and valves, increasing power consumption. To address the environmental concerns of conventional scale inhibitors, this paper explores biodegradable and eco-friendly alternatives. It examines the effects of organic additives on calcium (Ca) and magnesium (Mg) scaling in water vaporization. Batch experiments were conducted with potable water and various organic molecules (saponin, caffeine, tannic acid, dextran, citrus pectin, Ficoll 400, and Triton X-100). Saponin showed the highest calcium scale inhibition efficiency (60.9%) followed by caffeine (49.6%) and tannic acid (39.6%), while Ficoll 400, pectin, and Triton X-100 were less effective. For the magnesium scale, caffeine was the most effective (97.4%) followed by saponin (88.6%) and tannic acid (67.1%). Inhibition efficiencies for magnesium-containing scales were generally higher than those for calcium scales. Regarding the inhibition mechanisms, saponin, caffeine, dextran, and tannic acid adsorbed onto mineral crystal growth sites according to the Langmuir model, while pectin, Triton X-100, and Ficoll 400 formed complexes with Ca2+ and Mg2+ in solution. Needle-like aragonite was the predominant form of calcium carbonate (CaCO3) with the most additives, except tannic acid, which produced rhombohedral calcite, and caffeine, which promoted flower-like vaterite CaCO3 crystallites. Saponin, caffeine, tannic acid, and dextran are effective, biodegradable, and environmentally friendly inhibitors for mineral scaling.

Circadian rhythm disruption with high-fat diet impairs glycemic control and bone quality.

Biological functions, including glycemic control and bone metabolism, are highly influenced by the body's internal clock. Circadian rhythms are biological rhythms that run with a period close to 24hours and receive input from environmental stimuli, such as the light/dark cycle. We investigated the effects of circadian rhythm disruption (CRD), through alteration of the light/dark schedule, on glycemic control and bone quality of mice. Ten-week-old male mice (C57/BL6, n=48) were given a low-fat diet (LFD) or a high-fat diet (HFD) and kept on a dayshift or altered schedule (RSS3) for 22weeks. Mice were divided into four experimental groups (n=12/group): Dayshift/LFD, Dayshift/HFD, RSS3/LFD, and RSS3/HFD. CRD in growing mice fed a HFD resulted in a diabetic state, with a 36.2% increase in fasting glucose levels compared to the Dayshift/LFD group. Micro-CT scans of femora revealed a reduction in inner and outer surface expansion for mice on a HFD and altered light schedule. Cancellous bone demonstrated deterioration of bone quality as trabecular number and thickness decreased while trabecular separation increased. While HFD increased cortical bone mineral density, its combination with CRD reduced this phenomenon. The growth of mineral crystals, determined by small angle X-ray scattering, showed HFD led to smaller crystals. Considering modifications of the organic matrix, regardless of diet, CRD exacerbated the accumulation of fluorescent advanced glycation end-products (fAGEs) in collagen. Strength testing of tibiae showed that CRD mitigated the higher strength in the HFD group and increased brittleness indicated by lower post-yield deflection and work-to-fracture. Consistent with accumulation of fAGEs, various measures of toughness were lowered with CRD, but combination of CRD with HFD protected against this decrease. Differences between strength and toughness results represent different contributions of structural and material properties of bone to energy dissipation. Collectively, these results demonstrate that combination of CRD with HFD impairs glycemic control and have complex effects on bone quality.

Evolution of Pyrite Compositions at the Sizhuang Gold Deposit, Jiaodong Peninsula, Eastern China: Implications for the Genesis of Jiaodong-Type Orogenic Gold Mineralization

Gold deposits in the Jiaodong Peninsula represent a primary gold resource in China and mostly exhibit similar ore-forming features related to sericite-quartz-pyrite alteration and other controls from (micro-)structural deformation. This study investigates the pyrite textures and trace elements in the Sizhuang gold deposit (>100 t Au) to document the key factors impacting on the genesis of the Jiaodong-type orogenic deposits. Three main types of pyrite are identified: (1) the first generation of pyrite (Py1) occurs as disseminated euhedral to subhedral grains in K-feldspar-albite-rutile-hematite and sericite alteration (stage 1), (2) Py2 as aggregates in quartz-sericite-pyrite altered rocks or quartz-pyrite veins (stage 2) can be subdivided into Py2a as irregular cores, Py2b as a zoned overgrowth on Py2a, and Py2c as overgrowth on early pyrite, and (3) Py3 as fine-grained crystals in siderite-polymetallic veins (stage 3). Primary gold at the Sizhuang deposit is coevally or slightly later deposited with Py2b, Py2c, and Py3. Laser ablation–inductively coupled plasma mass spectrometry (LA–ICP–MS) analyses show that the highest Co and Ni contents in Py1 and high but variable Co in Py2b favors the involvement of deep high-temperature magmatic waters at stage 1 and middle stage 2. The elevated As contents from Py2a to Py2c and depletion of trace elements (e.g., Co, Ni, As and Te) and high Au/Co, Cu/Ni, and As/Ni values in Py2a and Py3, combined with published H-O isotope data, imply a meteoric water ingress during stage 2–3. Thus, the fluid evolution at Sizhuang is a consequence of pulsed deep magmatic fluid release plus progressive meteoric fluid ingress. The rhythmic Co–As–Ni–Au bands of Py2b additionally suggest episodic changes in the composition of ore-forming fluids. Moreover, the sharp textural features (e.g., pyrite overgrowth on previously cataclastic crystals) of Py2 and As-Cu-rich and Co-poor bands in zoned Py2b probably also reflect rapid metal deposition and self-organization and subsequent mineral crystal growth due to the pressure release during phase separation in the Sizhuang deposit. Considering the significantly concentrated gold (>1300 t) in the regional Jiaojia fault zone and Au-bearing mineral formation related to phase separation (boiling) in the Sizhuang deposit, gold mineralization in the Sizhuang deposit was interpreted to be controlled by the pressure-driver owing to the seismic activities in the Jiaojia fault system.

Mineral Crystal Thickness in Calcified Cartilage and Subchondral Bone in Healthy and Osteoarthritic Human Knees.

ABSTRACTOsteoarthritis (OA) is the most common joint disease, where articular cartilage degradation is often accompanied with sclerosis of the subchondral bone. However, the association between OA and tissue mineralization at the nanostructural level is currently not understood. In particular, it is technically challenging to study calcified cartilage, where relevant but poorly understood pathological processes such as tidemark multiplication and advancement occur. Here, we used state‐of‐the‐art microfocus small‐angle X‐ray scattering with a 5‐μm spatial resolution to determine the size and organization of the mineral crystals at the nanostructural level in human subchondral bone and calcified cartilage. Specimens with a wide spectrum of OA severities were acquired from both medial and lateral compartments of medial compartment knee OA patients (n = 15) and cadaver knees (n = 10). Opposing the common notion, we found that calcified cartilage has thicker and more mutually aligned mineral crystals than adjoining bone. In addition, we, for the first time, identified a well‐defined layer of calcified cartilage associated with pathological tidemark multiplication, containing 0.32 nm thicker crystals compared to the rest of calcified cartilage. Finally, we found 0.2 nm thicker mineral crystals in both tissues of the lateral compartment in OA compared with healthy knees, indicating a loading‐related disease process because the lateral compartment is typically less loaded in medial compartment knee OA. In summary, we report novel changes in mineral crystal thickness during OA. Our data suggest that unloading in the knee might be involved with the growth of mineral crystals, which is especially evident in the calcified cartilage. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

In Vitro Study on the Effect of a New Bioactive Desensitizer on Dentin Tubule Sealing and Bonding.

Bioactive mineral-based dentin desensitizers that can quickly and effectively seal dentinal tubules and promote dentin mineralization are desired. This in vitro study evaluated a novel nanohydroxyapatite-based desensitizer, Predicta (PBD, Parkell), and its effect on bond strength of dental adhesives. Human dentin discs (2-mm thick) were subjected to 0.5 M EDTA to remove the smear layer and expose tubules, treated with PBD, and processed for surface and cross-sectional SEM examination before and after immersion in simulated body fluid (SBF) for four weeks (ISO 23317-2014). The effects of two dental desensitizers on the microshear bond strength of a universal adhesive and a two-step self-etch system were compared. SEM showed coverage and penetration of nanoparticles in wide tubules on the PBD-treated dentin at the baseline. After four weeks in SBF, untreated dentin showed amorphous mineral deposits while PBD-treated dentin disclosed a highly mineralized structure integrated with dentin. Desensitizers significantly reduced microshear bond strength test (MSBS) of adhesives by 15–20% on average, depending on the bonding protocol. In conclusion, PBD demonstrated effective immediate tubules sealing capability and promoted mineral crystal growth over dentin and into the tubules during SBF-storage. For bonding to desensitizer-treated dentin, a two-step self-etching adhesive or universal bond with phosphoric acid pretreatment are recommended.

Mineral Scale Prevention on Electrically Conducting Membrane Distillation Membranes Using Induced Electrophoretic Mixing.

The growth of mineral crystals on surfaces is a challenge across multiple industrial processes. Membrane-based desalination processes, in particular, are plagued by crystal growth (known as scaling), which restricts the flow of water through the membrane, can cause membrane wetting in membrane distillation, and can lead to the physical destruction of the membrane material. Scaling occurs when supersaturated conditions develop along the membrane surface due to the passage of water through the membrane, a process known as concentration polarization. To reduce scaling, concentration polarization is minimized by encouraging turbulent conditions and by reducing the amount of water recovered from the saline feed. In addition, antiscaling chemicals can be used to reduce the availability of cations. Here, we report on an energy-efficient electrophoretic mixing method capable of nearly eliminating CaSO4 and silicate scaling on electrically conducting membrane distillation (ECMD) membranes. The ECMD membrane material is composed of a percolating layer of carbon nanotubes deposited on porous polypropylene support and cross-linked by poly(vinyl alcohol). The application of low alternating potentials (2 Vpp,1Hz) had a dramatic impact on scale formation, with the impact highly dependent on the frequency of the applied signal, and in the case of silicate, on the pH of the solution.

Cholesterol Regulates the Incorporation and Catalytic Activity of Tissue-Nonspecific Alkaline Phosphatase in DPPC Monolayers.

Matrix vesicles (MVs) are a special class of extracellular vesicles that drive bone and dentin mineralization by providing the essential enzymes and ions for the nucleation and propagation of mineral crystals. Tissue-nonspecific alkaline phosphatase (TNAP) is an integral protein of MV membrane and participates in biomineralization by hydrolyzing extracellular pyrophosphate (PPi), a strong mineralization inhibitor, and forming inorganic phosphate (Pi), necessary for the growth of mineral crystals inside MVs and their propagation once released in the extracellular matrix. MV membrane is enriched in cholesterol (CHOL), which influences the incorporation and activity of integral proteins in biologic membranes; however, how CHOL controls the incorporation and activity of TNAP in MV membrane has not yet been elucidated. In the present study, Langmuir monolayers were used as a MV membrane biomimetic model to assess how CHOL affects TNAP incorporation and activity. Surface pressure-area (π-A) isotherms of binary dipalmitoilphosphatidylcholine (DPPC)/CHOL monolayers showed that TNAP incorporation increases with CHOL concentration. Infrared spectroscopy showed that CHOL influences the conformation and orientation of the enzyme. Optical-fluorescence micrographs of the monolayers revealed the tendency of TNAP to incorporate into CHOL-rich microdomains. These data suggest that TNAP penetrates more efficiently and occupies a higher surface area into monolayers with a lower CHOL concentration due to the higher membrane fluidity. However, the quantity of enzyme transferred to solid supports as well as the enzymatic activity were higher using monolayers with a higher CHOL concentration due to increased rigidity that changes the enzyme orientation at the air-solid interface. These data provide new insights regarding the interfacial behavior of TNAP and CHOL in MVs and shed light on the biochemical and biophysical processes occurring in the MV membrane during biomineralization at the molecular level.

The impact of metastasis on the mineral phase of vertebral bone tissue

The negative impact of metastases on the mechanical performance of vertebral bone is often attributed to reduced bone density and/or compromised architecture. However limited characterization has been done on the impact of metastasis on the mineralization of bone tissue and resulting changes in material behaviour. This study aimed to evaluate the impact of metastasis on micro and nano scale characteristics of the mineral phase of bone, specifically mineral crystal growth, homogeneity of mineralization and changes in intrinsic material properties. Female athymic rats were inoculated with HeLa or Ace-1 cancer cells lines producing osteolytic or mixed (osteolytic & osteoblastic) metastases respectively (N=17 per group). A maximum of 21 days was allowed between inoculation and sacrifice of inoculated rats and healthy age-matched uninoculated controls (N=11). X-ray diffraction was used to assess average crystal size in crushed L1-L3 vertebrae; backscatter electron microscopy and nanoindentation were utilized to evaluate modifications in bone mineral density distribution and material behaviour (tissue hardness and modulus) in sagittal-sectioned, embedded and polished L5 vertebrae. HeLa inoculated samples showed reduced mineral crystal width compared to healthy controls. While both types of metastatic involvement reduced tissue mineral content, pathological osteoblastic bone, specific to Ace-1 inoculated samples, significantly decreased tissue mineral homogeneity, whereas osteolytic bone from HeLa samples saw a slight increase in homogeneity. The modulus and hardness of pathological osteoblastic bone was diminished compared to all other bone. Elucidating changes in material behaviour and mineralization of bone tissue is key to defining bone quality in the presence of metastatic involvement.

The supramolecular structure of bone: X-ray scattering analysis and lateral structure modeling.

The evolution of vertebrates required a key development in supramolecular evolution: internally mineralized collagen fibrils. In bone, collagen molecules and mineral crystals form a nanocomposite material comparable to cast iron in tensile strength, but several times lighter and more flexible. Current understanding of the internal nanoscale structure of collagen fibrils, derived from studies of rat tail tendon (RTT), does not explain how nucleation and growth of mineral crystals can occur inside a collagen fibril. Experimental obstacles encountered in studying bone have prevented a solution to this problem for several decades. This report presents a lateral packing model for collagen molecules in bone fibrils, based on the unprecedented observation of multiple resolved equatorial reflections for bone tissue using synchrotron small-angle X-ray scattering (SAXS; ∼1 nm resolution). The deduced structure for pre-mineralized bone fibrils includes features that are not present in RTT: spatially discrete microfibrils. The data are consistent with bone microfibrils similar to pentagonal Smith microfibrils, but are not consistent with the (nondiscrete) quasi-hexagonal microfibrils reported for RTT. These results indicate that collagen fibrils in bone and tendon differ in their internal structure in a manner that allows bone fibrils, but not tendon fibrils, to internally mineralize. In addition, the unique pattern of collagen cross-link types and quantities in mineralized tissues can be can be accounted for, in structural/functional terms, based on a discrete microfibril model.

Cloning and mineralization-related functions of the calponin gene in Chlamys farreri

Calponin is a widely distributed protein which is associated with the bio-mineralization process in vertebrates. Recently, a new type of calponin has been found in shellfish; the present study aimed to determine if this gene in shellfish functions in bio-mineralization, one of the most important processes in a mollusk's growth. We chose Chlamys farreri, a seashell species with great economic value, as the object of the study and obtained its full-length cDNA to study the function of calponin by gene expression analysis, shell notching experiment and RNA interference assays. Calponin in C. farreri is a basic protein that is highly conserved among mollusk species. Except for high expression in the adductor muscle and foot, which correlated with its function of regulating muscle contraction, the calponin gene was expressed more in the mantle than in other tissues. The expression of the gene was induced by shell notching and an RNA interference assay showed that inhibition of calponin expression caused the growth of irregular mineral crystals on the shell. Further analysis indicated that calponin might function by regulating the expression of other mineralization-related genes. Calponin is a mineralization-related protein in C. farreri that might influence mineral crystal growth by affecting the expressions of other proteins, such as matrix proteins and mineralization-regulating proteins.

RO membrane mineral scaling in the presence of a biofilm

Abstract The influence of a pre-existing biofilm on RO membrane mineral scaling was evaluated using gypsum as a model scalant. The biofilm was established using microfiltered secondary treated wastewater effluent on-site in a wastewater treatment facility. Mineral scaling was then monitored via direct visual observation of crystal growth on the membrane surface in a transparent plate-and-frame RO cell. SEM imaging of the membrane surface and sectioned biofilm revealed gypsum crystals within the biofilm matrix, including the protrusion of crystal rods from within the biofilm. Both mineral scale surface coverage and crystal number density were greater in the presence of the biofilm. Moreover, individual mineral crystal growth rate within the biofilm was significantly higher relative to growth in the absence of a biofilm. Analyses of crystal growth and nucleation rates, within the biofilm and in its absence, suggest concentration polarization enhancement within the biofilm. The present study suggests that mineral scaling may not be restricted to tail elements in RO plants, but could also occur in lead elements where biofilms can enhance concentration polarization. Therefore, understanding of the coupling of biofouling and mineral scaling may be of particular significance to the operation and design of RO plants in water reuse applications.

Control over the Vertical Growth of Single Calcitic Crystals in Biomineralized Structures

Acidic biomacromolecules frequently incorporate into biomineralized structures to control the morphology and extent of crystal growth. The study of such processes has been hindered by the scarcity of a model system that mimics the influence of acidic biomacromolecules on mineral crystal growth. A carbonic anhydrase-assisted system was developed to model CaCO3 deposition at an air/solution interface. Textured CaCO3 crystals were found to grow in a direction orthogonal (vertical) to the air/solution interface. The crystal growth anisotropy became more pronounced upon addition of an anionic polymer, and an amorphous morphology was found at sufficiently high polymer concentrations. X-ray diffraction and high-resolution transmission electron microscopy studies showed that most calcite crystals grew along the (014) and (001) planes vertically, whereas the (012) and (110) planes were oriented in the lateral direction. The added acidic polymers adsorbed predominantly onto the (012) or (110) faces of the growing ...

Nanoscale Imaging of Mineral Crystals inside Biological Composite Materials Using X-Ray Diffraction Microscopy

We for the first time applied x-ray diffraction microscopy to the imaging of mineral crystals inside biological composite materials--intramuscular fish bone--at the nanometer scale resolution. We identified mineral crystals in collagen fibrils at different stages of mineralization. Based on the experimental results and biomineralization analyses, we suggested a dynamic model to account for the nucleation and growth of mineral crystals in the collagen matrix. The results obtained from this study not only further our understanding of the complex structure of bone, but also demonstrate that x-ray diffraction microscopy will become an important tool to study biological materials.

Amelogenin expression in long bone and cartilage cells and in bone marrow progenitor cells

The amelogenin protein is considered as the major molecular marker of developing ectodermal enamel. Recent data suggest other roles for amelogenin beyond structural regulation of enamel mineral crystal growth. Here we describe our novel discovery of amelogenin expression in long bone cells, in cartilage cells, in cells of the epiphyseal growth plate, and in bone marrow stromal cells.

Amelogenin, a major structural protein in mineralizing enamel, is also expressed in soft tissues: brain and cells of the hematopoietic system

The amelogenin protein is considered as the major molecular marker of developing and mineralizing ectodermal enamel. It regulates the shape, size, and direction of growth of the enamel mineral crystallite. Recent data suggest other roles for amelogenin beyond regulation of enamel mineral crystal growth. The present study describes our recent discovery of amelogenin expression in soft tissues: in brain and in cells of the hematopoietic system, such as macrophages, megakaryocytes and in some of the hematopoietic stem cells. Reverse transcription-polymerase chain reaction (RT-PCR) followed by cDNA sequencing revealed, in mouse brain, two amelogenin mRNA isoforms: the full-length amelogenin including exon 4, and the isoform lacking exon 4. Immunohistochemistry revealed amelogenin expression in brain glial cells. Mouse macrophages were found to express the full-length amelogenin sequence lacking exon 4. Confocal microscopy revealed colocalization of amelogenin and CD41 (a megakaryocyte marker), as well as amelogenin and CD34 (a hematopoietic stem cell marker) in some of the bone marrow cells. The expression of amelogenin, a major structural protein of the mineralizing extracellular enamel matrix, also in cells of non-mineralizing soft tissues, suggests that amelogenin is multifunctional. Several different potential functions of amelogenin are discussed.

Aging of microstructural compartments in human compact bone.

Composition of microstructural compartments in compact bone of aging male subjects was assessed using Raman microscopy. Secondary mineralization of unremodeled fragments persisted for two decades. Replacement of these tissue fragments with secondary osteons kept mean composition constant over age, but at a fully mineralized limit. Slowing of remodeling may increase fracture susceptibility through an increase in proportion of highly mineralized tissue. In this study, the aging process in the microstructural compartments of human femoral cortical bone was investigated and related to changes in the overall tissue composition within the age range of 17-73 years. Raman microprobe analysis was used to assess the mineral content, mineral crystallinity, and carbonate substitution in fragments of primary lamellar bone that survived remodeling for decades. Tissue composition of the secondary osteonal population was investigated to determine the composition of turned over tissue volume. Finally, Raman spectral analysis of homogenized tissue was performed to evaluate the effects of unremodeled and newly formed tissue on the overall tissue composition. The chemical composition of the primary lamellar bone exhibited two chronological stages. Organic matrix became more mineralized and the crystallinity of the mineral improved during the first stage, which lasted for two decades. The mineral content and the mineral crystallinity did not vary during the second stage. The results for the primary lamellar bone demonstrated that physiological mineralization, as evidenced by crystal growth and maturation, is a continuous process that may persist as long as two decades, and the growth and maturation process stops after the organic matrix becomes "fully mineralized." The average mineral content and the average mineral crystallinity of the homogenized tissue did not change with age. It was also observed that the mineral content of the homogenized tissue was consistently greater than the osteons and similar to the "fully mineralized" stage of primary bone. The results of this study demonstrated that unremodeled compartments of bone grow older through maturation and growth of mineral crystals in a protracted fashion. However, the secondary osteonal remodeling impedes this aging process and maintains the mean tissue age fairly constant over decades. Therefore, slowing of remodeling may lead to brittle bone tissue through accumulation of fully mineralized tissue fragments.

Osteopontin deficiency increases mineral content and mineral crystallinity in mouse bone.

Fourier transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) were used to characterize the mineral in bones of two different lines of Opn-deficient (Opn-/-) mice and their background-matched wild-type controls (Opn+/+). Sections of tibia and femur from 12-week-old and 16-week-old mice were evaluated with a spatial resolution between 10 microm (FTIRM) and 7 microm (FTIRI). FTIRI was used to examine 400 microm x 400 microm areas in cortical bone and trabecular bone and FTIRM examined selected 20 microm x 20 microm areas at sites within these anatomically defined areas. Despite the absence of an obvious phenotype in Opn-deficient mice, being undetectable by radiographic and histological methods, FTIRM analyses revealed that the relative amount of mineral in the more mature areas of the bone (central cortical bone) of Opn-knockout mice was significantly increased. Moreover, mineral maturity (mineral crystal size and perfection) throughout all anatomic regions of the Opn-deficient bone was significantly increased. The 2-dimensional, color-coded data (images) produced by FTIRI showed similar increases in mineral maturity in the Opn-/- bone, however, the crystallinity parameters were less sensitive, and significance was not achieved in all areas analyzed. Nonetheless, the findings of increased mineral content and increased crystal size/perfection in both lines of Opn-deficient mice at both ages are consistent with in vitro data indicating that Opn is a potent inhibitor of mineral formation and mineral crystal growth and proliferation, and also support a role for Opn in osteoclast recruitment and function.

Physicochemical and microscopical study of calcific deposits from natural and bioprosthetic heart valves. Comparison and implications for mineralization mechanism.

Natural and bioprosthetic heart valves suffer from calcification, despite their differences in etiology and tissue material. The mechanism of developing calcific deposits in valve tissue is still not elucidated. The calcific deposits developed on human natural and bioprosthetic heart valves have been investigated and compared by physicochemical studies and microscopy investigations and the results were correlated with possible mechanisms of mineral crystal growth. Deposits from 16 surgically excised calcified valves (seven natural aortic and nine bioprosthetic porcine aortic valves) were examined by chemical analysis, FTIR, XRD, and SEM-EDS. The Ca/P molar ratio of the deposits from bioprosthetic valves (1.52+/-0.06) was significantly lower compared to that of the natural valves (1.83+/-0.03) (p=0.05, 1-way ANOVA). SEM-EDS examination of the two types of valve deposits revealed the coexistence of large (>20 microm) and medium (5-20 microm) plate-like crystals as well as microcrystalline (<5 microm) calcium phosphate mineral formations. The results confirmed the hypothesis that the mineral salt of calcified valves is a mixture of calcium phosphate phases such as dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP) and hydroxyapatite (HAP). DCPD and OCP are suggested to be precursor phases transformed to HAP by hydrolysis. The lower value of the Ca/P molar ratio found in the bioprostheses, in comparison with that corresponding in natural valves, was ascribed to the higher content in these deposits in precursor phases DCPD and OCP which were subsequently transformed into HAP. On the basis of chemical composition of the deposits and their morphology it is suggested that crystal growth proceeds in both types of valves by the same mechanism (hydrolysis of precursor phases to HAP) in spite of their differences in etiology, material, and possible initiation pathways.

The primary calcification in bones follows removal of decorin and fusion of collagen fibrils.

To elucidate the mechanisms of primary calcification in bone, ultrastructural changes in collagen fibrils, as well as cytochemical alteration of proteoglycan, especially decorin, were investigated morphologically in 19-day postcoitum embryonic rat calvariae. Below the osteoblast layer, calcification of the osteoid area increased in direct proportion to its distance from the osteoblasts. In the uncalcified osteoid area, collagen fibrils near matrix vesicles possessed sharp contours and were a uniform 50 nm in diameter. Immunoelectron microscopy revealed decorin to be abundantly localized in the vicinity of the collagen fibrils. In the osteoid area undergoing the process of calcification, collagen fibrils tended to fuse side by side. Where calcification was progressed, this fusion was even more so. Some very large fibrils exhibited complicated contours, 400 nm or more in diameter. Although the calcification at this stage affected areas both inside and outside of the collagen fibrils, the interior areas manifested a lower density of calcification. The immunolocalization of decorin was also much decreased around these fibrils. Thus, primary calcification in bone matrix follows the removal of decorin and fusion of collagen fibrils. This phenomenon may aid in the process of calcification and bone formation, because (1) inhibitors of calcification, such as decorin, are removed, (2) the fusion of collagen fibrils provides the room necessary for rapid growth of mineral crystals, and (3) the soft elastic bone matrix containing abundant fused collagen fibrils less subjective to calcification is safe for both maternal and embryonic bodies and is convenient for subsequent bone remodeling.

Expression of osteopontin, a urinary inhibitor of stone mineral crystal growth, in rat kidney

Cultured mouse kidney cortical cells secrete osteopontin, a bone matrix protein that is also found in urine. Osteopontin is associated with cell proliferation/tumerogenesis and also inhibits kidney stone mineral crystal growth [1]. Using antibodies raised against osteopontin isolated from the culture medium, we localized osteopontin in normal rat kidney. Fluorescence, confocal and electron microscopy revealed osteopontin primarily in cells of the descending thin limb of the loop of Henle (DTL) and in papillary surface epithelium (PSE) in the area of the calyceal fornix. In situ hybridization with labeled RNA made from a cDNA that contains the entire coding sequence for mouse osteopontin revealed message at the same sites at which protein was demonstrated by immunocytochemistry. Immunogold labeling was localized to a population of dense vesicles distinct from lysosomes and endosomes. To examine the turnover of osteopontin, rats were injected with the protein synthesis inhibitor cyclohexamide, 14 mg/kg, six hours prior to kidney fixation. These kidneys no longer demonstrated osteopontin in DTL and the immunofluorescence in the papillary surface was attenuated. Thus, osteopontin is secreted at two sites in the kidney where urine is highly concentrated in stone mineral constituents. It has a relatively rapid turnover, suggesting that it could be subject to physiological regulation. Osteopontin may be important in the normal endogenous defense against kidney stone formation.