Formation Of Hydroxyapatite Crystals Research Articles

Lactoferrin network with MC3T3-E1 cell proliferation, auxiliary mineralization, antibacterial functions: A multifunctional coating for biofunctionalization of implant surfaces

Developing biocompatible, low-immunoreactive, and antibacterial implants are challenging yet fundamental to osteosynthesis. In this study, mineralization-stimulative and antibacterial networking nanostructures are assembled via amyloid-like aggregation of lactoferrin (LF) triggered by reducing the intramolecular disulfide bonds. Due to the adhesive property of their rich β-sheet architecture, the LF networks are amenable to the deposition upon the surface of various implant materials, functionalizing the implants with cell-proliferative, mineralization-stimulative, and antibacterial properties. Specifically, the abundant functional groups and amino acids exposed on the surface of LF networks provide abundant functional microdomains for subsequent mineralization of different forms of calcium ions and promote the formation of hydroxyapatite (HAp) crystals in simulated body fluids. We further demonstrate that the LF network inherits the innate antibacterial properties of LF and exerts a synergistic antibacterial ability with surface-enriched positively charged and hydrophobic amino acid residues, disrupting bacterial biofilm formation, enhancing microbial cell wall perturbation, and ultimately leading to microbial death. The results underscore the feasibility of the LF network as a multifunctional coating on bioscaffold surfaces, which may provide insight into its future applications in next-generation artificial bone implants with bacterial/biofilm clearance and bone tissue remodeling capabilities.

CaPO4-Mediated CKD of Crystallo-Tubular-Nephropathy [CKD-CTN]—A Crystal and Nanotube-Induced Geo-Environmental Disease

ImportanceMore than forty million people from certain tropical countries are at risk of developing a non-conventional form of chronic kidney disease (CKD), CKD of multifactorial etiology (CKDmfo). This is also known as CKD of unknown etiology (CKDu). Worldwide, it kills more than 20,000 people annually.FindingsCKDmfo is a chronic tubulointerstitial renal disease caused by groundwater-induced hydroxy- and fluorapatite nano-crystals and nano-tube formation in renal tubules and cortical tissues, in conjunction with chronic intravascular volume depletion, chronic renal anaemia, and ischemia. To manifest this gradually developing renal failure, consuming hard water having, higher concentrations of calcium, phosphates, and fluoride for more than 10 years is necessary. The disease progresses when the kidneys fail to repair or due to ongoing renal tissue damage in the presence of micronutrient deficiency, chronic dehydration, renal ischemia, chronic inflammation, and oxidative stress. These prevents regenerations of renal tissues.ConclusionConsumption of stagnant groundwater concentrated with ions, like Ca2+, PO4, and F− due to prolonged annual droughts over many years, in conjunction with lesser water intake and chronic dehydration, creates a conducive internal milieu for CaPO4 crystallisation in renal tissues. This proposed primary etiology of the crystal-tubular-nephropathy (CTN) provides an insight into a deeper understanding of the use of cost-effective strategies for prevention, early intervention, and eradication of CKD-CTN. In addition to the nano-crystal/nano-tube concept, we provide supporting scientific evidence that Mg2+ in water and the diet does not promote the disease; instead, it prevents crystal formation and developing CKDmfo, as with some other CKDs.RelevanceWe present novel CaPO4 hydroxyapatite crystal formation concepts in a hyperosmolar fluid in renal tissues that causes CKDmfo. Besides, the protective (e.g., magnesium) and enhancing (e.g., dehydration, drinking stagnant hard water) mechanisms that cause CKD-CTN are explored. A new understanding of causative mechanisms paves a path for cost-effective targeted interventions to prevent and eliminate CKDmfo. These principles apply to all CKDmfo/CKDu-affected countries to protect the renal health of farm labourers and others who regularly engage in physical work in hot and dry environments. Providing affordable potable water, increasing water consumption, and avoiding harmful behaviours are critical measures for eliminating CKDmfo.

Effect of glycerol and H3PO4 on the bioactivity and degradability of rod-like SBA-15 particles with active surface for bone tissue engineering applications

In the quest to increase the quality of life and life expectancy, there is a great demand to develop advanced medical devices capable of facing current health challenges. Santa Barbara Amorphous (SBA-15) mesoporous silica has been demonstrated to be a remarkable option for a variety of biomedical applications, from tissue engineering to drug delivery, due to its unique hierarchical porous structure. This work reports on the effect of using different ratios and concentrations of an acid mixture mediated by HCl/H 3 PO 4 that in combination with a glycerol-modified silane precursor favoured obtaining bioactive SBA-15 mesoporous silica rods. The surfactant was removed by solvent extraction which allowed to keep a substantial amount of silanol groups (SiOH) at the surface. All the evaluated samples showed homogeneous rod shape and particle size, as well as high bioactivity after being immersed for 14 days in simulated body fluid (SBF). Different structural arrangements of needle-type carbonate hydroxyapatite (HCA) crystals were produced from SBA-15 upon immersion in SBF. By scanning electron microscopy (SEM) characterization, it was observed that rod-shaped SBA-15 materials displayed higher degradability when the concentration of H 3 PO 4 was increased. An indirect cell viability assay with MC3T3-E1 osteoblast precursor cells was performed by incubating cells in cell culture medium exposed to SBA-15 particles for 1 and 4 days. The results obtained from the WST-8 assay and fluorescent staining showed no negative effect on cell viability, indicating a lack of cytotoxicity of the rod-like SBA-15 particles. • Solvent extraction allowed keeping a large amount of silanol groups at the surface. • Higher the concentration of H 3 PO 4 smaller the size of the SBA-15 rods. • PO 4 3− species and SiOH promoted the formation of carbonate hydroxyapatite crystals. • The use of H 3 PO 4 and glycerol facilitated the degradation process at body pH. • The species released from SBA-15 particles after 4 days boosted cell proliferation.

Aesculetin Accelerates Osteoblast Differentiation and Matrix-Vesicle-Mediated Mineralization.

The imbalance between bone resorption and bone formation in favor of resorption results in bone loss and deterioration of bone architecture. Osteoblast differentiation is a sequential event accompanying biogenesis of matrix vesicles and mineralization of collagen matrix with hydroxyapatite crystals. Considerable efforts have been made in developing naturally-occurring plant compounds, preventing bone pathologies, or enhancing bone regeneration. Coumarin aesculetin inhibits osteoporosis through hampering the ruffled border formation of mature osteoclasts. However, little is known regarding the effects of aesculetin on the impairment of matrix vesicle biogenesis. MC3T3-E1 cells were cultured in differentiation media with 1–10 μM aesculetin for up to 21 days. Aesculetin boosted the bone morphogenetic protein-2 expression, and alkaline phosphatase activation of differentiating MC3T3-E1 cells. The presence of aesculetin strengthened the expression of collagen type 1 and osteoprotegerin and transcription of Runt-related transcription factor 2 in differentiating osteoblasts for 9 days. When ≥1–5 μM aesculetin was added to differentiating cells for 15–18 days, the induction of non-collagenous proteins of bone sialoprotein II, osteopontin, osteocalcin, and osteonectin was markedly enhanced, facilitating the formation of hydroxyapatite crystals and mineralized collagen matrix. The induction of annexin V and PHOSPHO 1 was further augmented in ≥5 μM aesculetin-treated differentiating osteoblasts for 21 days. In addition, the levels of tissue-nonspecific alkaline phosphatase and collagen type 1 were further enhanced within the extracellular space and on matrix vesicles of mature osteoblasts treated with aesculetin, indicating matrix vesicle-mediated bone mineralization. Finally, aesculetin markedly accelerated the production of thrombospondin-1 and tenascin C in mature osteoblasts, leading to their adhesion to preformed collagen matrix. Therefore, aesculetin enhanced osteoblast differentiation, and matrix vesicle biogenesis and mineralization. These findings suggest that aesculetin may be a potential osteo-inductive agent preventing bone pathologies or enhancing bone regeneration.

Carotid Artery Calcification: What We Know So Far.

Carotid artery calcification (CAC) is a well-known marker of atherosclerosis and is linked to a high rate of morbidity and mortality. CAC is divided into two types: intimal and medial calcifications, each with its own set of risk factors. Vascular calcification is now understood to be an active, enzymatically regulated process involving dystrophic calcification and endothelial dysfunction at an early stage. This causes a pathogenic inflammatory response, resulting in calcium phosphate deposition in the form of microcalcifications, which causes plaque formation, ultimately becoming unstable with sequelae of complications. If the inflammation goes away, hydroxyapatite crystal formation takes over, resulting in macro-calcifications that help to keep the plaque stable. As CAC can be asymptomatic, it is critical to identify it early using diagnostic imaging.The carotid artery calcification score is calculated using computed tomography angiography (CTA), which is a confirmatory test that enables the examination of plaque composition and computation of the carotid artery calcification score. Magnetic resonance angiography (MRA), which is sensitive as CTA, duplex ultrasound (DUS), positron emission tomography, and computed tomography (PET-CT) imaging with (18) F-Sodium Fluoride, and Optical Coherence Tomography (OCT) are some of the other diagnostic imaging modalities used. The current therapeutic method starts with the best medical care and is advised for all CAC patients. Carotid endarterectomy and carotid stenting are two treatment options that have mixed results in terms of effectiveness and safety. When patient age and anatomy, operator expertise, and surgical risk are all considered, the agreement is that both techniques are equally beneficial.

Mild Reaction of Highly-Oriented Collagen Fibril Arrays with Simulated Body Fluid

The highly-oriented collagen fibrils that paralleled to one (rubbing) direction were fabricated by which the collagen molecular solution was spin-coated and self-assembled on the rubbed polyimide film. Subsequently, the hydroxyapatite crystals were precipitated on the collagen fibrils by immersing into simulated body fluid. In details, the carboxyl groups on the collagen fibrils were used as a reaction field for adsorption of Ca2+ ions and promoted the formation of hydroxyapatite crystals. As a result, the hydroxyapatite crystals grew along the a-axis leading to the formation of stable interfaces between hydroxyapatite crystals and collagen fibrils. Moreover, the oriented collagen fibril arrays were more useful for the nucleation and growth of hydroxyapatite. Therefore, we successfully fabricated the highly-oriented collagen fibril arrays which were useful for the precipitation of hydroxyapatite crystals.

A time-course Raman spectroscopic analysis of spontaneous in vitro microcalcifications in a breast cancer cell line

Microcalcifications are early markers of breast cancer and can provide valuable prognostic information to support clinical decision-making. Current detection of calcifications in breast tissue is based on X-ray mammography, which involves the use of ionizing radiation with potentially detrimental effects, or MRI scans, which have limited spatial resolution. Additionally, these techniques are not capable of discriminating between microcalcifications from benign and malignant lesions. Several studies show that vibrational spectroscopic techniques are capable of discriminating and classifying breast lesions, with a pathology grade based on the chemical composition of the microcalcifications. However, the occurrence of microcalcifications in the breast and the underlying mineralization process are still not fully understood. Using a previously established model of in vitro mineralization, the MDA-MB-231 human breast cancer cell line was induced using two osteogenic agents, inorganic phosphate (Pi) and β-glycerophosphate (βG), and direct monitoring of the mineralization process was conducted using Raman micro-spectroscopy. MDA-MB-231 cells cultured in a medium supplemented with Pi presented more rapid mineralization (by day 3) than cells exposed to βG (by day 11). A redshift of the phosphate stretching peak for cells supplemented with βG revealed the presence of different precursor phases (octacalcium phosphate) during apatite crystal formation. These results demonstrate that Raman micro-spectroscopy is a powerful tool for nondestructive analysis of mineral species and can provide valuable information for evaluating mineralization dynamics and any associated breast cancer progression, if utilized in pathological samples.The mineralization of MDA-MB-231 breast cells was investigated using Raman micro-spectroscopy. Mineralization was induced by two osteogenic agents: inorganic phosphate (Pi) and β-Glycerophosphate (βG). The results show that the uptake of Pi allows a faster mineralization and the uptake of βG indicated the presence of a precursor phase during the hydroxyapatite crystal formation.

Self-assembly and mineralization of full-length human amelogenin and its functional fragments in vitro.

To investigate the dynamic process of the self-assembly behaviors of a full-length human amelogenin (AM) and its functional fragments tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide(LRAP) in vitro and its role in hydroxyapatite (HA) crystal formation. The full-length human AM and its functional fragments, TRAP and LRAP, were reassembled and purified in vitro. The protein solution of 100 µg‧mL-1, pH=8, was prepared in Tris-HCl and incubated at room temperature for 1-15 min. Their self-assembly behaviors were observed and compared under a transmission electron microscope (TEM). The full-length AM was added to artificial saliva and incubated for 3 days. A scanning electron microscope (SEM) was used in observing the morphology of the induced new crystals. Then, TARP and LRAP were added. The resulting solution was incubated for 3 days and then observed again. When pH=8, the full-length human AM and TRAP assembly started spontaneously and formed "nanospheres" after 15 min.The nanospheres formed by TRAP existed independently, with a uniform size but without obvious internal structures. The full-length AM was assembled hierarchically, which formed "nanospheres" and further extended in all directions, formed a chain structure, and then aggregated into a net. The self-assembly behavior of LRAP was not obvious. Proteins mostly existed in the form of monomers without "nanosphere" formation. Only few oligomers were observed. The full-length AM was induced independently for 3 days to form rod-shaped HA crystals. TRAP and LRAP proteins were added, after 3 days the crystal elongation was obvious in the c axis, but the growth in plane A and plane B was poor. The self-assembly and mineralization behaviors of full-length human AM, TRAP, and LRAP were consistent with the directional growth mechanism of HA crystals in vivo, providing a theoretical basis for the role of the fragments in the growth and maturation of HA crystals.

Bioactive Glass Modified Calcium Phosphate Cement with Improved Bioactive Properties: A Potential Material for Dental Pulp-Capping Approaches

Direct pulp capping (DPC) is one of the treatment plans for deep caries with mechanical pulp exposure that can replace invasive treatments. This study aimed to assess the apatite-forming ability and solubility of a calcium phosphate cement (CPC) modified with bioactive glass (BG) as a potential bioactive material for DPC.Three different biomaterials including CPC, BG, and CPC/BG composite were used in this study. For bioactivity evaluation, specimens were immersed in simulated body fluid (SBF) for 5 time periods (3, 7, 14, 21 and 28 days). The samples were analyzed by SEM, EDS and XRD to confirm the formation of hydroxyapatite. The solubility was calculated by measuring the initial and final mass according to the ISO 6876 specifications.According to the results of this study, SEM observations and XRD analysis revealed higher formation of hydroxyapatite crystals in the CPC/BG Group and also at the shorter time than those in the CPC and BG groups. Concerning solubility, the CPC group showed the most solubility after 7 days and the BG group showed the lowest one. At this time the difference between CPC and BG groups was statistically meaningful (p value=0.003). After 30 days the CPC/BG group exhibited the lowest solubility value. At the day 30, the CPC and BG groups showed significant difference in their solubility (p value=0.04).).Based on the results, addition of BG to CPC improved bioactivity properties of CPC material and did not affect its solubility adversely. The CPC/BG composite seems to be a promising material for DPC. Further in vivo studies are needed to prove its clinical success.

Three-Dimensional Culture of Ameloblast-Originated HAT-7 Cells for Functional Modeling of Defective Tooth Enamel Formation.

Background: Amelogenesis, the formation of dental enamel, is well understood at the histomorphological level but the underlying molecular mechanisms are poorly characterized. Ameloblasts secrete enamel matrix proteins and Ca2+, and also regulate extracellular pH as the formation of hydroxyapatite crystals generates large quantities of protons. Genetic or environmental impairment of transport and regulatory processes (e.g. dental fluorosis) leads to the development of enamel defects such as hypomineralization. Aims: Our aims were to optimize the culture conditions for the three-dimensional growth of ameloblast-derived HAT-7 cells and to test the effects of fluoride exposure on HAT-7 spheroid formation. Methods: To generate 3D HAT-7 structures, cells were dispersed and plated within a Matrigel extracellular matrix scaffold and incubated in three different culture media. Spheroid formation was then monitored over a two-week period. Ion transporter and tight-junction protein expression was investigated by RT-qPCR. Intracellular Ca2+ and pH changes were measured by microfluorometry using the fluorescent dyes fura-2 and BCECF. Results: A combination of Hepato-STIM epithelial cell differentiation medium and Matrigel induced the expansion and formation of 3D HAT-7 spheroids. The cells retained their epithelial cell morphology and continued to express both ameloblast-specific and ion transport-specific marker genes. Furthermore, like two-dimensional HAT-7 monolayers, the HAT-7 spheroids were able to regulate their intracellular pH and to show intracellular calcium responses to extracellular stimulation. Finally, we demonstrated that HAT-7 spheroids may serve as a disease model for studying the effects of fluoride exposure during amelogenesis. Conclusion: In conclusion, HAT-7 cells cultivated within a Matrigel extracellular matrix form three-dimensional, multi-cellular, spheroidal structures that retain their functional capacity for pH regulation and intracellular Ca2+ signaling. This new 3D model will allow us to gain a better understanding of the molecular mechanisms involved in amelogenesis, not only in health but also in disorders of enamel formation, such as those resulting from fluoride exposure.

A novel investigation on characterization of bioactive glass cement and chitosan-gelatin membrane for jawbone tissue engineering

The aim of this work was to investigate the characterization of a bioactive glass (BG) ceramic prepared by sol–gel technique and chitosan-gelatin membranes prepared by lyophilization technique containing 0, 1 and 2 vol.% ethanol. Early-absorbing and late-absorbing bone substitutes in practice constitute the main volume of bone substitutes used by dentist and orthopedic surgeons. When the graft is to be gradually replaced by normal bone, the important issue is the duration and rate of graft absorption. The prepared samples were characterized using fourier transformed infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis. The presence of Ag nanoparticles into bioactive glass was evaluated. Porous membranes were examined under scanning electron microscopy (SEM) to estimate the size of the pores and analysis the morphological behavior. The pore diameter could be controlled within the range 10–30 µm by adjusting the percent of ethanol. The formation of needle-like hydroxyapatite (HA) crystals on the surface of the membrane after 7 days immersion in the ringer's solution was also assessed using SEM images. The SEM images results illustrated the porous structures in the membranes. The average pore size for chitosan-gelatin membrane with 2% ethanol were 30±6.3 μm. Finally, these obtained results suggested that the developed membrane with 2% ethanol possess the prerequisites for tissue engineering and can be used for jawbone tissue engineering applications.

Bioactive alginate/carrageenan/calcium silicate porous scaffolds for bone tissue engineering

Porous bioactive alginate/carrageenan/calcium silicate scaffolds for bone tissue engineering were fabricated. The scaffolds were prepared by dispersing the synthesized calcium silicate in an aqueous solution of alginate and carrageenan at 90 °C. The scaffolds were shaped by freeze-drying and further crosslinked by 0.5, 1.0 and 1.5 M CaCl2 for 60 and 120 min. The scaffolds crosslinked by 1.5 M CaCl2 for 120 min achieved the highest in vitro dimension stability. The formation of hydroxyapatite crystals was observed on the scaffolds surface after soaking in simulated body fluid (SBF) at 37 °C for 7–28 days, indicating in vitro bioactivity of the scaffolds. The presence of calcium silicate could enhance not only the bioactivity, but also the mechanical properties of the scaffolds comparable to the cancellous bone. Moreover, the dimension and mechanical properties of the wet scaffolds could recover to the original after four cycles of mechanical testing at 50 % strain. The scaffolds were nontoxic to human living cells, in which the in vitro drug release behavior of the scaffold using diclofenac as a model drug was suitable for the treatment of acute inflammation after surgery. Therefore, these scaffolds were considered to be the candidate materials for bone replacement.

Effect of Hydroxyapatite Composite Nano-Artificial Bone on Treatment and Rehabilitation of Patients with Ankle Joint Injury.

The formation of natural bone tissue is the result of the joint regulation of multiple template molecules. Although its complex hierarchical structure has been studied for many years, the mechanism of biomineralization of hard bone tissue has not been fully clarified. In this paper, the nanocomposites obtained by mineralization were characterized and analyzed, and the effect of the template on the crystal formation of hydroxyapatite was studied. The characterization results show that the main phase of the inorganic mineral obtained by template mineralization is the hydroxyapatite phase. The nano-apatite composite particles with an inorganic component content of 90.2% have the highest loading efficiency, reaching 67.9 mg/g. By statistical analysis of the pain scores at 5 days, 10 days, and 15 days after ankle injury, it was found that the average pain score of the treatment group was smaller than that of the control group. Two weeks later, the clinical efficacy judgment standard statistics show that the treatment group has a 22.5% improvement in healing rate, a significant increase in 3.18%, and a total effective rate of 8.71%, which is significantly better than the control group.

Phosphorus recovery via the formation of hydroxyapatite crystals at various nitrogen loading rate in an anammox-based UAFB

A strategy that integrates the anammox and hydroxyapatite crystallization in an up-flow anaerobic fixed-bed reactor (UAFB) was investigated to simultaneously remove nitrogen and recover phosphorus. During the 430 days of operation, 73.1 ± 6.6% of influent phosphorus was removed with an efficient nitrogen removal efficiency of 87.8 ± 1.7%. After long-term operation, numerous acicular and micron-sized crystals were observed on the matured biofilm, of which the phosphorus content was around 10.21% (wt%) and hydroxyapatite was the main form of crystals through SEM-EDS, FT-IR and XRD analysis. The variation of substrates along the axial length of UAFB showed that phosphate removal was positively correlated with anammox and pH. Moreover, three anammox bacteria including Candidatus Brocadia (19.73%), Candidatus Jettenia (0.49%) and Candidatus Kuenenia (0.85%) were detected at the bottom of UAFB, while Candidatus Jettenia (4.67%) was dominant at the top. Hence, the anammox-based biofilm system could be alternative for the recovery of phosphorus from nutrient-rich wastewater.

Poly lactic acid-akermanite composite scaffolds prepared by fused filament fabrication for bone tissue engineering

In this study, Poly Lactic Acid (PLA) scaffolds containing different amounts of akermanite (Ak) particles were prepared using the fused filament fabrication (FFF) technology. Ak particles were fabricated using sol–gel method. Field Emission Scanning Electron Microscopy (FESEM) and Transmission Electron Microscopy (TEM) images revealed that the size of the synthesized Ak particles was 100 ± 20 nm. The structure of the 3D printed scaffolds was investigated by Scanning Electron Microscopy (SEM) and X-ray diffraction (XRD). Distribution of the particles was characterized by Energy Dispersive Spectroscopy (EDS: map), showing the uniform distribution of particles through the PLA matrix. The results of the compression test showed that the strength (plateau stress) of the scaffolds was considerably improved by increasing the Ak particles from 27 ± 2 MPa for the neat polymer scaffold to 45 ± 3 MPa for PLA-30% Ak scaffold, which is near to the values of human cortical bone. The results of the bioactivity test (immersing the scaffolds in the Simulated Body Fluid (SBF) for 7, 14, 21 and 28 days) also indicated the enhanced formation of hydroxyapatite crystals on the scaffold surface upon increasing the immersion time and the Ak particles. While, the increment of Ak particles up to 20 wt.% had no significant effect on printability of scaffolds, and after that, reduced the scaffold printability, so the PLA-Ak 20 wt.% scaffold can be considered as a promising candidate for bone tissue engineering.

Self Assembling Peptide P11-4 for Enamel Remineralization: A Biomimetic Approach

Treatment of caries through conventional modalities involves an invasive approach of excavation of the carious lesion followed by a restoration. This compromises on the strength and integrity of the tooth structure. Minimal invasive dentistry aims at early detection of the carious lesion and their remineralization. Self assembling peptides find its application in the medical field due to its ability to form a scaffold through a process of hierarchical organization into nano structures. Self-assembling peptide P11-4 is a novel technology for enamel remineralization through a biomimetic approach. The technology simulates normal enamel histogenesis. It induces de novo precipitation of hydroxyapatite crystals by forming a three dimensional scaffold matrix. The formation of the scaffold involves the conversion of the peptide from a low viscosity fluid to a nematic gel under low pH conditions. This scaffold matrix further attracts calcium ions and leads to formation of hydroxyapatite crystals. Being minimally invasive, it helps in preserving the strength and integrity of the tooth structure. The aim of the article is to review the technology of self assembling peptides for enamel regeneration and its potential as a material for successful treatment of early carious lesions through a minimally invasive approach.

Mechanism of action of SNF472, a novel calcification inhibitor to treat vascular calcification and calciphylaxis.

No therapy is approved for vascular calcification or calcific uraemic arteriolopathy (calciphylaxis), which increases mortality and morbidity in patients undergoing dialysis. Deposition of hydroxyapatite (HAP) crystals in arterial walls is the common pathophysiologic mechanism. The mechanism of action of SNF472 to reduce HAP deposition in arterial walls was investigated. We examined SNF472 binding features (affinity, release kinetics and antagonism type) for HAP crystals in vitro, inhibition of calcification in excised vascular smooth muscle cells from rats and bone parameters in osteoblasts from dogs and rats. SNF472 bound to HAP with affinity (KD ) of 1-10 μM and saturated HAP at 7.6 μM. SNF472 binding was fast (80% within 5 min) and insurmountable. SNF472 inhibited HAP crystal formation from 3.8 μM, with complete inhibition at 30.4 μM. SNF472 chelated free calcium with an EC50 of 539 μM. Chelation of free calcium was imperceptible for SNF472 1-10 μM in physiological calcium concentrations. The lowest concentration tested in vascular smooth muscle cells, 1 μM inhibited calcification by 67%. SNF472 showed no deleterious effects on bone mineralization in dogs or in rat osteoblasts. These experiments show that SNF472 binds to HAP and inhibits further HAP crystallization. The EC50 for chelation of free calcium is 50-fold greater than a maximally effective SNF472 dose, supporting the selectivity of SNF472 for HAP. These findings indicate that SNF472 may have a future role in the treatment of vascular calcification and calcific uraemic arteriolopathy in patients undergoing dialysis.

Biocompatibility of biphasic α,β-tricalcium phosphate ceramics in vitro

The biocompatibility of biphasic α,β-tricalcium phosphate ceramics, obtained by annealing a compact preform based on β-tricalcium phosphate powder, was studied in vitro. It was found that within 10–30 days the adhesion of primary dental pulp stem cells located on the surface of biphasic α,β-tricalcium phosphate ceramics is suppressed. Decrease of the cell number on the surface of biphasic α,β-tricalcium phosphate ceramics, most likely, can be associated with both the pH level (acidic) as a result of hydrolysis of the more soluble phase of α-tricalcium phosphate and with the nature of surface that changes as a result of the formation and growth of hydroxyapatite crystals.

Hyperphosphatemia with low FGF7 and normal FGF23 and sFRP4 levels in the circulation characterizes pediatric hypophosphatasia.

Hypophosphatasia (HPP) is the inborn-error-of-metabolism caused by loss-of-function mutation(s) of the ALPL gene that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). TNSALP in healthy individuals is on cell surfaces richly in bone, liver, and kidney. Thus, TNSALP natural substrates accumulate extracellularly in HPP, including inorganic pyrophosphate (PPi), a potent inhibitor of hydroxyapatite crystal formation and growth. Superabundance of extracellular PPi (ePPi) in HPP impairs mineralization of bones and teeth, often leading to rickets during childhood and osteomalacia in adult life and to tooth loss at any age. HPP's remarkably broad-ranging severity is largely explained by nearly four hundred typically missense mutations throughout the ALPL gene that are transmitted as an autosomal dominant or autosomal recessive trait. In the clinical laboratory, the biochemical hallmark of HPP is low serum ALP activity (hypophosphatasemia). However, our experience indicates that hyperphosphatemia from increased renal reclamation of filtered inorganic phosphate (Pi) is also common. Herein, from our prospective single-center study, we document throughout the clinical spectrum of non-lethal pediatric HPP that hyperphosphatemia reflects increased renal tubular threshold maximum for phosphorus adjusted for the glomerular filtration rate (TmP/GFR). To explore its pathogenesis, we studied mineral metabolism and quantitated circulating levels of three phosphatonins [fibroblast growth factor 23 (FGF23), secreted frizzled-related protein 4 (sFRP4), and fibroblast growth factor 7 (FGF7)] in 41 pediatric patients with HPP, 73 with X-linked hypophosphatemia (XLH), and 15 healthy pediatric control (CTR) subjects. The HPP and XLH cohorts had normal serum total and ionized calcium and parathyroid hormone levels (Ps > 0.10) and uncompromised glomerular filtration. In XLH, serum FGF23 was characteristically elevated (P < 0.0001) and despite hypophosphatemia sFRP4 was normal (P > 0.4) while FGF7 was low (P < 0.0001). In HPP, despite hyperphosphatemia serum FGF23 and sFRP4 were normal (Ps > 0.8) while FGF7 was low (P < 0.0001). Subsequently, in rats, we confirmed that FGF7 is phosphaturic. Thus, hyperphosphatemia in non-lethal pediatric HPP is associated with phosphatonin insufficiency together with, as we discuss, ePPi excess and diminished renal TNSALP activity.

Tissue-nonspecific alkaline phosphatase promotes the osteogenic differentiation of osteoprogenitor cells

Tissue-nonspecific alkaline phosphatase (TNAP) is expressed in the calcification sites of the skeletal tissue. It promotes hydroxyapatite crystal formation by degrading inorganic pyrophosphate (PPi) and increasing inorganic phosphate (Pi) concentration. However, abnormalities in Alpl−/− mouse-derived osteoblasts are poorly understood, and the involvement of TNAP in osteoblast differentiation remains unclear. Therefore, in this study, we aimed to investigate the precise role of TNAP in osteoblast differentiation. TNAP inhibition by levamisole, a reversible TNAP inhibitor, suppressed the expression of osteoblast differentiation marker genes in wild-type osteoblastic cells. Alpl overexpression increased the expression of master osteoblast transcription factor genes runt-related transcription factor 2 (Runx2) and Sp7 and the mature osteoblast and osteocyte marker genes, bone γ-carboxyglutamate protein 2 (Bglap2) and dentin matrix protein 1 (Dmp1), respectively in Alpl-deficient osteoblastic cells. TNAP regulated Runx2 expression, which in turn regulated the expression of all other osteoblast markers, except Dmp1. Dmp1 expression was independent of RUNX2 but was dependent on extracellular Pi concentration in Runx2-deficient osteogenic cells. These results suggest that TNAP functions as an osteogenic differentiation regulator either by regulating Runx2 expression or by controlling extracellular Pi concentration.