Calcium phosphate controls nucleation and growth of calcium oxalate crystal phases in kidney stones.

Kidney stone disease is a serious disease due to the severe pain it causes, high morbidity, and high recurrence rate. Notably, calcium oxalate stones are the most common type of kidney stone. Calcium oxalate appears in two forms in kidney stones: the stable phase, monohydrate (COM), and the metastable phase, dihydrate (COD). Particularly, COM stones with concentric structures are hard and difficult to treat. However, the factor determining the growth of either COM or COD crystals in the urine, which is supersaturated for both phases, remains unclear. This study shows that calcium phosphate ingredients preferentially induce COM crystal nucleation and growth, by observing and analyzing kidney stones containing both COM and COD crystals. The forms of calcium phosphate are not limited to Randall's plaques (1-2 mm size aggregates, which contain calcium phosphate nanoparticles and proteins, and form in the renal papilla). For example, aggregates of strip-shaped calcium phosphate crystals and fields of dispersed calcium phosphate microcrystals (nano to micrometer order) also promote the growth of concentric COM structures. This suggests that patients who excrete urine with a higher quantity of calcium phosphate crystals may be more prone to forming hard and troublesome COM stones.

Ecofriendly solidification of sand using microbially induced calcium phosphate precipitation.

This study introduces microbiologically induced calcium phosphate precipitation (MICPP) as a novel and environmentally sustainable method of soil stabilization. Using Limosilactobacillus sp., especially NBRC 14511 and fish bone solution (FBS) extracted from Tuna fish bones, the study was aimed at testing the feasibility of calcium phosphate compounds (CPCs) deposition and sand stabilization. Dynamic changes in pH and calcium ion (Ca2+) concentration during the precipitation experiments affected the precipitation and sequential conversion of dicalcium phosphate dihydrate (DCPD) to hydroxyapatite (HAp), which was confirmed by XRD and SEM analysis. Sand solidification experiments demonstrated improvements in unconfined compressive strength (UCS), especially at higher Urea/Ca2+ ratios. The UCS values obtained were 10.35 MPa at a ratio of 2.0, 3.34 MPa at a ratio of 1.0, and 0.43 MPa at a ratio of 0.5, highlighting the advantages of MICPP over traditional methods. Microstructural analysis further clarified the mineral composition, demonstrating the potential of MICPP in environmentally friendly soil engineering. The study highlights the promise of MICPP for sustainable soil stabilization, offering improved mechanical properties and reducing environmental impact, paving the way for novel geotechnical practices.

Osteopontin stabilization and collagen containment slows amorphous calcium phosphate transformation during human aortic valve leaflet calcification.

Calcification of aortic valve leaflets is a growing mortality threat for the 18 million human lives claimed globally each year by heart disease. Extensive research has focused on the cellular and molecular pathophysiology associated with calcification, yet the detailed composition, structure, distribution and etiological history of mineral deposition remains unknown. Here transdisciplinary geology, biology and medicine (GeoBioMed) approaches prove that leaflet calcification is driven by amorphous calcium phosphate (ACP), ACP at the threshold of transformation toward hydroxyapatite (HAP) and cholesterol biomineralization. A paragenetic sequence of events is observed that includes: (1) original formation of unaltered leaflet tissues: (2) individual and coalescing 100's nm- to 1 µm-scale ACP spherules and cholesterol crystals biomineralizing collagen fibers and smooth muscle cell myofilaments; (3) osteopontin coatings that stabilize ACP and collagen containment of nodules preventing exposure to the solution chemistry and water content of pumping blood, which combine to slow transformation to HAP; (4) mm-scale nodule growth via ACP spherule coalescence, diagenetic incorporation of altered collagen and aggregation with other ACP nodules; and (5) leaflet diastole and systole flexure causing nodules to twist, fold their encasing collagen fibers and increase stiffness. These in vivo mechanisms combine to slow leaflet calcification and establish previously unexplored hypotheses for testing novel drug therapies and clinical interventions as viable alternatives to current reliance on surgical/percutaneous valve implants.

Ca(H2PO4)2 and MgSO4 activated nitrogen-related bacteria and genes in thermophilic stage of compost.

This study was conducted to investigate the effects of Ca(H2PO4)2 and MgSO4 on the bacterial community and nitrogen metabolism genes in the aerobic composting of pig manure. The experimental treatments were set up as control (C), 1% Ca(H2PO4)2 + 2% MgSO4 (CaPM1), and 1.5% Ca(H2PO4)2 + 3% MgSO4 (CaPM2), which were used at the end of composting for potting trials. The results showed that Ca(H2PO4)2 and MgSO4 played an excellent role in retaining nitrogen and increasing the alkali-hydrolyzed nitrogen (AN), available phosphorus (AP), and available potassium (AK) contents of the composts. Adding Ca(H2PO4)2 and MgSO4 changed the microbial community structure of the compost. The microorganisms associated with nitrogen retention were activated. The complexity of the microbial network was enhanced. Genetic prediction analysis showed that the addition of Ca(H2PO4)2 and MgSO4 reduced the accumulation of nitroso-nitrogen and the process of denitrification. At the same time, despite the reduction of genes related to nitrogen fixation, the conversion of ammonia to nitrogenous organic compounds was promoted and the stability of nitrogen was increased. Mantel test analysis showed that Ca(H2PO4)2 and MgSO4 can affect nitrogen transformation-related bacteria and thus indirectly affect nitrogen metabolism genes by influencing the temperature, pH, and organic matter (OM) of the compost and also directly affected nitrogen metabolism genes through PO43- and Mg2+. The pot experiment showed that composting with 1.5% Ca(H2PO4)2 + 3% MgSO4 produced the compost product that improved the growth yield and nutrient content of cilantro and increased the fertility of the soil. In conclusion, Ca(H2PO4)2 and MgSO4 reduces the loss of nitrogen from compost, activates nitrogen-related bacteria and genes in the thermophilic phase of composting, and improves the fertilizer efficiency of compost products. KEY POINTS: • Ca(H2PO4)2 and MgSO4 reduced the nitrogen loss and improved the compost effect • Activated nitrogen-related bacteria and altered nitrogen metabolism genes • Improved the yield and quality of cilantro and fertility of soil.

The Rise in Tubular pH during Hypercalciuria Exacerbates Calcium Stone Formation.

In calcium nephrolithiasis (CaNL), most calcium kidney stones are identified as calcium oxalate (CaOx) with variable amounts of calcium phosphate (CaP), where CaP is found as the core component. The nucleation of CaP could be the first step of CaP+CaOx (mixed) stone formation. High urinary supersaturation of CaP due to hypercalciuria and an elevated urine pH have been described as the two main factors in the nucleation of CaP crystals. Our previous in vivo findings (in mice) show that transient receptor potential canonical type 3 (TRPC3)-mediated Ca2+ entry triggers a transepithelial Ca2+ flux to regulate proximal tubular (PT) luminal [Ca2+], and TRPC3-knockout (KO; -/-) mice exhibited moderate hypercalciuria and microcrystal formation at the loop of Henle (LOH). Therefore, we utilized TRPC3 KO mice and exposed them to both hypercalciuric [2% calcium gluconate (CaG) treatment] and alkalineuric conditions [0.08% acetazolamide (ACZ) treatment] to generate a CaNL phenotype. Our results revealed a significant CaP and mixed crystal formation in those treated KO mice (KOT) compared to their WT counterparts (WTT). Importantly, prolonged exposure to CaG and ACZ resulted in a further increase in crystal size for both treated groups (WTT and KOT), but the KOT mice crystal sizes were markedly larger. Moreover, kidney tissue sections of the KOT mice displayed a greater CaP and mixed microcrystal formation than the kidney sections of the WTT group, specifically in the outer and inner medullary and calyceal region; thus, a higher degree of calcifications and mixed calcium lithiasis in the kidneys of the KOT group was displayed. In our effort to find the Ca2+ signaling pathophysiology of PT cells, we found that PT cells from both treated groups (WTT and KOT) elicited a larger Ca2+ entry compared to the WT counterparts because of significant inhibition by the store-operated Ca2+ entry (SOCE) inhibitor, Pyr6. In the presence of both SOCE (Pyr6) and ROCE (receptor-operated Ca2+ entry) inhibitors (Pyr10), Ca2+ entry by WTT cells was moderately inhibited, suggesting that the Ca2+ and pH levels exerted sensitivity changes in response to ROCE and SOCE. An assessment of the gene expression profiles in the PT cells of WTT and KOT mice revealed a safeguarding effect of TRPC3 against detrimental processes (calcification, fibrosis, inflammation, and apoptosis) in the presence of higher pH and hypercalciuric conditions in mice. Together, these findings show that compromise in both the ROCE and SOCE mechanisms in the absence of TRPC3 under hypercalciuric plus higher tubular pH conditions results in higher CaP and mixed crystal formation and that TRPC3 is protective against those adverse effects.

Sex-specific Stone-forming Phenotype in Mice During Hypercalciuria/Urine Alkalinization.

Sex differences in kidney stone formation are well known. Females generally have slightly acidic blood and higher urine pH when compared with males, which makes them more vulnerable to calcium stone formation, yet the mechanism is still unclear. We aimed to examine the role of sex in stone formation during hypercalciuria and urine alkalinization through acetazolamide and calcium gluconate supplementation, respectively, for 4 weeks in wild-type (WT) and moderately hypercalciuric [TRPC3 knockout [KO](-/-)] male and female mice. Our goal was to develop calcium phosphate (CaP) and CaP+ calcium oxalate mixed stones in our animal model to understand the underlying sex-based mechanism of calcium nephrolithiasis. Our results from the analyses of mice urine, serum, and kidney tissues show that female mice (WT and KO) produce more urinary CaP crystals, higher [Ca2+], and pH in urine compared to their male counterparts. We identified a sex-based relationship of stone-forming phenotypes (types of stones) in our mice model following urine alkalization/calcium supplementation, and our findings suggest that female mice are more susceptible to CaP stones under those conditions. Calcification and fibrotic and inflammatory markers were elevated in treated female mice compared with their male counterparts, and more so in TRPC3 KO mice compared with their WT counterparts. Together these findings contribute to a mechanistic understanding of sex-influenced CaP and mixed stone formation that can be used as a basis for determining the factors in sex-related clinical studies.

Anatomical Brushite-Coated Mg-Nd-Zn-Zr Alloy Cage Promotes Cervical Fusion: One-Year Results in Goats.

In this study, an anatomical brushite-coated Mg-Nd-Zn-Zr alloy cage was fabricated for cervical fusion in goats. The purpose of this study was to investigate the cervical fusion effect and degradation characteristics of this cage in goats. The Mg-Nd-Zn-Zr alloy cage was fabricated based on anatomical studies, and brushite coating was prepared. Forty-five goats were divided into three groups, 15 in each group, and subjected to C2/3 anterior cervical decompression and fusion with tricortical bone graft, Mg-Nd-Zn-Zr alloy cage, or brushite-coated Mg-Nd-Zn-Zr alloy cage, respectively. Cervical radiographs and computed tomography (CT) were performed 3, 6, and 12 months postoperatively. Blood was collected for biocompatibility analysis and Mg2+ concentration tests. The cervical spine specimens were obtained at 3, 6, and 12 months postoperatively for biomechanical, micro-CT, scanning electron microscopy coupled with energy dispersive spectroscopy, laser ablation-inductively coupled plasma-time-of-flight mass spectrometry, and histological analysis. The liver and kidney tissues were obtained for hematoxylin and eosin staining 12 months after surgery for biosafety analysis. Imaging and histological analysis showed a gradual improvement in interbody fusion over time; the fusion effect of the brushite-coated Mg-Nd-Zn-Zr alloy cage was comparable to that of the tricortical bone graft, and both were superior to that of the Mg-Nd-Zn-Zr alloy cage. Biomechanical testing showed that the brushite-coated Mg-Nd-Zn-Zr alloy cage achieved better stability than the tricortical bone graft at 12 months postoperatively. Micro-CT showed that the brushite coating significantly decreases the corrosion rate of the Mg-Nd-Zn-Zr alloy cage. In vivo degradation analysis showed higher Ca and P deposition in the degradation products of the brushite-coated Mg-Nd-Zn-Zr alloy cage, and no hyperconcentration of Mg was detected. Biocompatibility analysis showed that both cages were safe for cervical fusion surgery in goats. To conclude, the anatomical brushite-coated Mg-Nd-Zn-Zr alloy cage can promote cervical fusion in goats, and the brushite-coated Mg-Nd-Zn-Zr alloy is a potential material for developing absorbable fusion cages.

Extracellular histones promote calcium phosphate-dependent calcification in mouse vascular smooth muscle cells.

Vascular calcification, a major risk factor for cardiovascular events, is associated with a poor prognosis in chronic kidney disease (CKD) patients. This process is often associated with the transformation of vascular smooth muscle cells (VSMCs) into cells with osteoblast-like characteristics. Damage-associated molecular patterns (DAMPs), such as extracellular histones released from damaged or dying cells, are suspected to accumulate at calcification sites. To investigate the potential involvement of DAMPs in vascular calcification, we assessed the impact of externally added histones (extracellular histones) on calcium and inorganic phosphate-induced calcification in mouse VSMCs. Our study found that extracellular histones intensified calcification. We also observed that the histones decreased the expression of VSMC marker genes while simultaneously increasing the expression of osteoblast marker genes. Additionally, histones treated with DNase I, which degrades dsDNA, attenuated this calcification, compared with the non-treated histones, suggesting a potential involvement of dsDNA in this process. Elevated levels of dsDNA were also detected in the serum of CKD model mice, underlining its potential role in vascular calcification in CKD. Our findings suggest that extracellular histones could play a pivotal role in the vascular calcification observed in CKD.

Enhancing cranial defect repair in rats: investigating the effect of combining Total Flavonoids from Rhizoma Drynariae with calcium phosphate/collagen scaffolds.

OBJECTIVE: To investigate the therapeutic efficacy of total flavonoids of Rhizoma Drynariae (TFRD) in conjunction with a calcium phosphate/collagen scaffold for the repair of cranial defects in rats. METHODS: The subjects, rats, were segregated into four groups: Control, TFRD, Scaffold, and TFRD + Scaffold. Cranial critical bone defects, 5 mm in diameter, were artificially induced through precise drilling. Post-surgery, at intervals of 2, 4, and 8 weeks, micro-CT scans were conducted to evaluate the progress of skull repair. Hematoxylin-eosin and Masson staining techniques were applied to discern morphological disparities, and immunohistochemical staining was utilized to ascertain the expression levels of local osteogenic active factors, such as bone morphogenetic protein 2 (BMP-2) and osteocalcin (OCN). RESULTS: Upon examination at the 8-week mark, cranial defects in the Scaffold and TFRD + Scaffold cohorts manifested significant repair, with the latter group displaying only negligible foramina. Micro-CT examination unveiled relative to its counterparts, and the TFRD + Scaffold groups exhibited marked bone regeneration at the 4- and 8-week intervals. Notably, the TFRD + Scaffold group exhibited substantial bone defect repair compared to the TFRD and Scaffold groups throughout the entire observation period, while histomorphological assessment demonstrated a significantly higher collagen fiber content than the other groups after 2 weeks. Immunohistochemical analysis further substantiated that the TFRD + Scaffold had augmented expression of BMP-2 at 2, 4 weeks and OCN at 2 weeks relative to other groups. CONCLUSIONS: The synergistic application of TFRD and calcium phosphate/collagen scaffold has been shown to enhance bone mineralization, bone plasticity, and bone histomorphology especially during initial osteogenesis phases.

Particulate beta-tricalcium phosphate and hydroxyapatite doped with silver promote in vitro osteoblast differentiation in MC3T3-E1 cells.

BACKGROUND: Calcium phosphates including ß-tricalcium phosphate (ß-TCP) and hydroxyapatite (HAp) have been widely used for bone regeneration application because of their high osteoconductive activities. In addition, various kinds of inorganic ions enhance differentiation, proliferation, and mineralization of osteoblasts. However, information about the effects of silver-doped ß-TCP [ß-TCP (Ag)] and HAp [HAp (Ag)] particles on osteogenic differentiation is not available yet. OBJECTIVE: We focused on the impact of ß-TCP (Ag) and HAp (Ag) particles on the osteogenic differentiation of MC3T3-E1 osteoblast precursor cells. METHODS: MC3T3-E1 osteoblast precursor cells were pre-treated by ß-TCP (Ag) or HAp (Ag). And then the medium was changed to differentiation medium. Subsequently, osteoblast differentiation-related markers were determined. RESULTS: We found that treatment with ß-TCP (Ag) or HAp (Ag) particles increased alkaline phosphatase activity in MC3T3-E1 cells. Expression of osteoblast differentiation-related genes also increased after treatment with ß-TCP (Ag) or HAp (Ag) particles, a response thought to be regulated by zinc finger-containing transcription factor osterix. The ratio of the receptor activator of nuclear factor kappa-B ligand (RANKL) to osteoprotegerin (OPG) was decreased by ß-TCP (Ag) and HAp (Ag) particles. CONCLUSION: Silver doping of ß-TCP and HAp particles is effective for bone regeneration.

Reconsidering the role of albumin towards amorphous calcium phosphate-based calciprotein particles formation and stability from a physico-chemical perspective.

The formation of calciprotein particles (CPPs) in serum is a physiological phenomenon fundamental to prevent the rise of ectopic calcifications. CPPs are colloidal hybrid particles made of amorphous calcium phosphate stabilized by a protein, fetuin-A. Since albumin is the most abundant protein present in serum, we aimed at understanding if it plays a synergic action together with fetuin-A towards CPPs formation and stability. CPPs were prepared using a constant fetuin-A concentration (5 µM) and different concentrations of albumin (0-606 µM). The stability of CPPs, their crystallization and sedimentation were followed in situ by combining turbidimetry, precipitation analysis and dynamic light scattering. The morphology was investigated by scanning electron microscopy and cryo-transmission electron microscopy, while crystallinity was inspected by infrared spectroscopy. The effect of albumin on the amount of formed CPPs was also studied, as well as the amount of protein adsorbed on CPPs. We found that albumin is not able to prolong the lifetime of the amorphous phase, but it is very effective in delaying the sedimentation of CPPs after crystallization. Albumin also significantly decreases the amount and size of CPPs when present in their synthetic medium, likely playing a fundamental role in our organism together with fetuin-A towards the stabilization of CPPs.

Calcium phosphate microcrystallopathy as a paradigm of chronic kidney disease progression.

PURPOSE OF REVIEW: Calciprotein particles (CPP) are colloidal mineral-protein complexes mainly composed of solid-phase calcium phosphate and serum protein fetuin-A. CPP appear in the blood and renal tubular fluid after phosphate intake, playing critical roles in (patho)physiology of mineral metabolism and chronic kidney disease (CKD). This review aims at providing an update of current knowledge on CPP. RECENT FINDINGS: CPP formation is regarded as a defense mechanism against unwanted growth of calcium phosphate crystals in the blood and urine. CPP are polydisperse colloids and classified based on the density and crystallinity of calcium phosphate. Low-density CPP containing amorphous (noncrystalline) calcium phosphate function as an inducer of FGF23 expression in osteoblasts and a carrier of calcium phosphate to the bone. However, once transformed to high-density CPP containing crystalline calcium phosphate, CPP become cytotoxic and inflammogenic, inducing cell death in renal tubular cells, calcification in vascular smooth muscle cells, and innate immune responses in macrophages. SUMMARY: CPP potentially behave like a pathogen that causes renal tubular damage, chronic inflammation, and vascular calcification. CPP have emerged as a promising therapeutic target for CKD and cardiovascular complications.

Avoiding and allowing apatite precipitation in oxygenic photolithotrophs.

The essential elements Ca and P, taken up and used metabolically as Ca2+ and H2 PO4 - /HPO4 2- respectively, could precipitate as one or more of the insoluble forms calcium phosphate (mainly apatite) if the free ion concentrations and pH are high enough. In the cytosol, chloroplast stroma, and mitochondrial matrix, the very low free Ca2+ concentration avoids calcium phosphate precipitation, apart from occasionally in the mitochondrial matrix. The low free Ca2+ concentration in these compartments is commonly thought of in terms of the role of Ca2+ in signalling. However, it also helps avoids calcium phosphate precipitation, and this could be its earliest function in evolution. In vacuoles, cell walls, and xylem conduits, there can be relatively high concentrations of Ca2+ and inorganic orthophosphate, but pH and/or other ligands for Ca2+ , suggests that calcium phosphate precipitates are rare. However, apatite is precipitated under metabolic control in shoot trichomes, and by evaporative water loss in hydathodes, in some terrestrial flowering plants. In aquatic macrophytes that deposit CaCO3 on their cell walls or in their environment as a result of pH increase or removal of inhibitors of nucleation or crystal growth, phosphate is sometimes incorporated in the CaCO3 . Calcium phosphate precipitation also occurs in some stromatolites.

Functionalized calcium phosphate nanoparticles to direct osteoprotegerin to bone lesion sites in a medaka (Oryzias latipes) osteoporosis model.

Calcium phosphate (CaP) is the inorganic part of hard tissues, such as bone, teeth and tendons, and has a high biocompatibility and good biodegradability. Therefore, CaP nanoparticles functionalized with DNA encoding bone anabolic factors are promising carrier-systems for future therapeutic development. Here, we analysed CaP nanoparticles in a genetically modified medaka fish model, where osteoporosis-like lesions can be induced by transgenic expression of receptor activator of nuclear factor kappa-B ligand (Rankl). Rankl-transgenic medaka were used to visualize and understand effects of microinjected functionalized CaP nanoparticles during modulation of osteoclast activity in vivo. For this, we synthetized multi-shell CaP nanoparticles by rapid precipitation of calcium lactate and ammonium hydrogen phosphate followed by the addition of plasmid DNA encoding the osteoclastogenesis inhibitory factor osteoprotegerin-b (Opgb). An additional layer of poly(ethyleneimine) was added to enhance cellular uptake. Integrity of the synthesized nanoparticles was confirmed by dynamic light scattering, scanning electron microscopy and energy dispersive X-ray spectroscopy. Fluorescently labelled CaP nanoparticles were microinjected into the heart, trunk muscle or caudal fins of Rankl-transgenic medaka embryos that expressed fluorescent reporters in various bone cell types. Confocal time-lapse imaging revealed a uniform distribution of CaP nanoparticles in injected tissues and showed that nanoparticles were efficiently taken up by macrophages that subsequently differentiated into bone-resorbing osteoclasts. After Rankl induction, fish injected with Opg-functionalized nanoparticles showed delayed or absent degradation of mineralized matrix, i.e. a lower incidence of osteoporosis-like phenotypes. This is proof of principle that CaP nanoparticles can be used as carriers to efficiently deliver modulatory compounds to osteoclasts and block their activity.

Calcium-to-phosphorus releasing ratio affects osteoinductivity and osteoconductivity of calcium phosphate bioceramics in bone tissue engineering.

Calcium phosphate (Ca-P) bioceramics, including hydroxyapatite (HA), biphasic calcium phosphate (BCP), and beta-tricalcium phosphate (ß-TCP), have been widely used in bone reconstruction. Many studies have focused on the osteoconductivity or osteoinductivity of Ca-P bioceramics, but the association between osteoconductivity and osteoinductivity is not well understood. In our study, the osteoconductivity of HA, BCP, and ß-TCP was investigated based on the osteoblastic differentiation in vitro and in situ as well as calvarial defect repair in vivo, and osteoinductivity was evaluated by using pluripotent mesenchymal stem cells (MSCs) in vitro and heterotopic ossification in muscles in vivo. Our results showed that the cell viability, alkaline phosphatase activity, and expression of osteogenesis-related genes, including osteocalcin (Ocn), bone sialoprotein (Bsp), alpha-1 type I collagen (Col1a1), and runt-related transcription factor 2 (Runx2), of osteoblasts each ranked as BCP > ß-TCP > HA, but the alkaline phosphatase activity and expression of osteogenic differentiation genes of MSCs each ranked as ß-TCP > BCP > HA. Calvarial defect implantation of Ca-P bioceramics ranked as BCP > ß-TCP ≥ HA, but intramuscular implantation ranked as ß-TCP ≥ BCP > HA in vivo. Further investigation indicated that osteoconductivity and osteoinductivity are affected by the Ca/P ratio surrounding the Ca-P bioceramics. Thus, manipulating the appropriate calcium-to-phosphorus releasing ratio is a critical factor for determining the osteoinductivity of Ca-P bioceramics in bone tissue engineering.

Application of the mineral-binding protein fetuin-A for the detection of calcified lesions.

Rationale: Calcium plays an essential role in the biology of vertebrates. Calcium content in body fluids is maintained within a narrow physiologic range by feedback control. Phosphate is equally important for metabolism and is likewise controlled, albeit over a wider range. This results in a nearly supersaturated state of calcium phosphate in body liquids driving mineral precipitation in soft tissues, which is actively prevented by calcification inhibitors. The hepatic plasma protein fetuin-A is a circulating mineralization inhibitor regulating calcium phosphate crystal growth and calcified matrix metabolism. Ectopic mineralization is associated with many pathological conditions aggravating their outcome. Current diagnostic methods lack sensitivity towards microcalcifications representing the initial stages of the process. Given the irreversibility of established calcifications, novel diagnostic tools capable of detecting nascent calcium phosphate deposits are highly desirable. Methods: We designed fluorescent fusion proteins consisting of fetuin-A coupled to a green or red fluorescent protein derivate, mEmerald or mRuby3, respectively. The proteins were expressed in mammalian cell lines. Sequence optimization resolved folding issues and increased sensitivity of mineral binding. Chimeric proteins were tested for their ability to detect calcifications in cell cultures and tissue sections retrieved from calcification-prone mice. Results: We employed novel genetically labeled fetuin-A-based fluorescent proteins for the detection of ectopic calcifications. We show that fetuin-A-based imaging agents are non-toxic and suitable for live imaging of microcalcifications beyond the detection limit of conventional staining techniques. The ability of fetuin-A to preferentially bind nascent calcium phosphate crystals allowed the resolution of histopathological detail of early kidney damage that went previously undetected. Endogenous expression of fetuin-A fluorescent fusion proteins allowed extended live imaging of calcifying cells with unprecedented sensitivity and specificity. Conclusion: Ectopic microcalcifications represent a major clinical concern lacking effective diagnostic and treatment options. In this paper, we describe novel highly sensitive fetuin-A-based fluorescent probes for imaging microcalcifications. We show that fusion proteins consisting of a fetuin-A mineral binding moiety and a fluorescent protein are superior to the routine methods for detecting calcifications. They also surpass in continuous live cell imaging the chemically fluorescence labeled fetuin-A, which we established previously.

Tissue engineering at the dentin-pulp interface using human treated dentin scaffolds conditioned with DMP1 or BMP2 plasmid DNA-carrying calcium phosphate nanoparticles.

Hard dental tissue pathologies, such as caries, are conventionally managed through replacement by tooth-colored inert biomaterials. Tissue engineering provides novel treatment approaches to regenerate lost dental tissues based on bioactive materials and/or signaling molecules. While regeneration in the form of reparative dentin (osteo-dentin) is feasible, the recapitulation of the tubular microstructure of ortho-dentin and its special features is sidelined. This study characterized in vitro, and in vivo human EDTA-treated, freeze-dried dentin matrices (HTFD scaffolds) conditioned with calcium phosphate nanoparticles (NPs) bearing plasmids encoding dentinogenesis-inducing factors (pBMP2/NPs or pDMP1/NPs). The uptake and transfection efficiency of the synthesized NPs on dental pulp stem cells (DPSCs) increased in a concentration- and time-dependent manner, as evaluated qualitatively by confocal laser microscopy and transmission electron microscopy, and quantitatively by flow cytometry, while, in parallel, cell viability decreased. HTFD scaffolds conditioned with the optimal transfectability-to-viability concentration at 4 µg Ca/mL of each of the pBMP2/NPs or pDMP1/NPs preserved high levels of cell viability, evidenced by live/dead staining in vitro and caused no adverse reactions after implantation on C57BL6 mice in vivo. HTFD/NPs constructs induced rapid and pronounced odontogenic shift of the DPSCs, as evidenced by relevant gene expression patterns of RunX2, ALP, BGLAP, BMP-2, DMP-1, DSPP by real-time PCR, and acquirement of polarized meta-mitotic phenotype with cellular protrusions entering the dentinal tubules as visualized by scanning electron microscopy. Taken together, HTFD/NPs constitute a promising tool for customized reconstruction of the ortho-dentin/odontoblastic layer barrier and preservation of pulp vitality. STATEMENT OF SIGNIFICANCE: In clinical dentistry, the most common therapeutic approach for the reconstruction of hard dental tissue defects is the replacement by resin-based restorative materials. Even modern bioactive materials focus on reparative dentinogenesis, leading to amorphous dentin-bridge formation in proximity to the pulp. Therefore, the natural microarchitecture of tubular ortho-dentin is not recapitulated, and the sensory and defensive role of odontoblasts is sidelined. This study approaches the reconstruction at the dentin-pulp interface using a construct of human treated dentin (HTFD) scaffold and plasmid-carrying nanoparticles (NPs) encoding dentinogenic factors (DMP-1 or BMP-2) with excellent in vitro and in vivo properties. As a future perspective, the HTFD/NPs constructs could act as bio-fillings for personalized reconstruction of the dentin-pulp interface.

Unidirectional porous beta-tricalcium phosphate as a potential bone regeneration material for infectious bony cavity without debridement in pyogenic spondylitis.

An 81-year-old man was initially diagnosed with T11 osteoporotic vertebral fracture. The fractured vertebral body was filled with unidirectional porous beta-tricalcium phosphate (ß-TCP) granules, and posterior spinal fixation was conducted using percutaneous pedicle screws. However, the pain did not improve, the inflammatory response increased, and bone destructive changes extended to T10. The correct diagnosis was pyogenic spondylitis with concomitant T11 fragility vertebral fracture. Revision surgery was conducted 2 weeks after the initial surgery, the T10 and T11 pedicle screws were removed, and refixation was conducted. After the revision surgery, the pain improved and mobilization proceeded. The infection was suppressed by the administration of sensitive antibiotics. One month after surgery, a lateral bone bridge appeared at the T10/11 intervertebral level. This increased in size over time, and synostosis was achieved at 6 months. Resorption of the unidirectional porous ß-TCP granules was observed over time and partial replacement with autologous bone was evident from 6 months after the revision surgery. Two years and 6 months after the revision surgery, although there were some residual ß-TCP and bony defect in the center of the vertebral body, the bilateral walls have well regenerated. This suggested that given an environment of sensitive antibiotic administration and restricted local instability, unidirectional porous ß-TCP implanted into an infected vertebral body may function as a resorbable bone regeneration scaffold without impeding infection control even without debridement of the infected bony cavity.

Basic calcium phosphate crystals induce the expression of extracellular matrix remodelling enzymes in tenocytes.

OBJECTIVES: Basic calcium phosphate (BCP) crystals contribute to several syndromes associated with tendon disease, including acute calcific tendinitis and Milwaukee shoulder syndrome. Interactions between BCP crystals and tenocytes (tendon cells) may contribute to these clinical syndromes. This study aimed to determine the direct effects of BCP crystals on tenocyte function and viability. METHODS: In vitro assays were used to assess changes in human tenocytes cultured with BCP crystals. Real-time PCR was used to determine changes in the expression of tendon-related genes and extracellular matrix remodelling enzymes (MMPs; a disintegrin and metalloproteases, ADAMTS; and tissue inhibitor of metalloproteinases, TIMPs). ELISA was used to measure protein concentrations in tenocyte supernatants. MTT and alamarBlue™ assays were used to determine changes in cell viability. RESULTS: BCP crystals upregulated tenocyte gene expression of MMP-1, MMP-3, ADAMTS-4 and TIMP-1 after 24 h. Time-course experiments showed expression peaked at 8 h for TIMP-1 and 48 h for MMP-1 and ADAMTS-4. Cyclooxygenase (COX)-1 gene expression was upregulated after 48 h. Tenocytes did not alter expression of scleraxis and tendon collagens, and expression of pro-inflammatory cytokines was not induced with BCP crystals. BCP crystals increased tenocyte release of prostaglandin E2 (PGE2) and MMP-1 protein after 24 h. However, neither COX-1 inhibition nor COX-2 inhibition led to consistent change in BCP crystal-induced tenocyte gene expression of extracellular matrix remodelling enzymes. BCP crystals had no effect on tenocyte viability. CONCLUSION: BCP crystals induce extracellular matrix remodelling enzymes, but not inflammatory cytokines, in tenocytes.

Effect of inoculation density of bone marrow mesenchymal stem cells cultured on calcium phosphate cement scaffold on osteogenic differentiation.

BACKGROUND: Calcium phosphate cements (CPCs) are biocompatible materials that have been evaluated as scaffolds in bone tissue engineering. At present, the stem cell density of inoculation on CPC scaffold varies. OBJECTIVE: The aim of this study is to analyze the effect of seeding densities on cell growth and osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) on a calcium phosphate cements (CPCs) scaffold. METHODS: BMMSCs derived from minipigs were seeded onto a CPC scaffold at three densities [1 million/mL (1M), 5 million/mL (5M) and 25 million/mL 25M)], and cultured for osteogenic induction for 1, 4 and 8 days. RESULTS: Well adhered and extended BMMSCs on the CPC scaffold showed significantly different proliferation rates within each seeding density group at different time points (P < 0.05). The number of live cells per unit area in 1M, 5M and 25M increased by 3.5, 3.9 and 2.5 folds respectively. The expression of ALP peaked at 4 days post inoculation with the fold-change being 2.6 and 2.8 times higher in 5M and 25M respectively as compared to 1M. The expression levels of OC, Coll-1 and Runx-2 peaked at 8 days post inoculation. CONCLUSIONS: An optimal seeding density may be more conducive for cell proliferation, differentiation, and extracellular matrix synthesis on scaffolds. We suggest the optimal seeding density should be 5 million/mL.