Na+ controls hypoxic signalling by the mitochondrial respiratory chain.

All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1-4, a phenomenon that occurs in hypoxia4-8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of the inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism.

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.

Recombinant human enamelin produced in Escherichia coli promotes mineralization in vitro.

BACKGROUND: Enamelin is an enamel matrix protein that plays an essential role in the formation of enamel, the most mineralized tissue in the human body. Previous studies using animal models and proteins from natural sources point to a key role of enamelin in promoting mineralization events during enamel formation. However, natural sources of enamelin are scarce and with the current study we therefore aimed to establish a simple microbial production method for recombinant human enamelin to support its use as a mineralization agent. RESULTS: In the study the 32 kDa fragment of human enamelin was successfully expressed in Escherichia coli and could be obtained using immobilized metal ion affinity chromatography purification (IMAC), dialysis, and lyophilization. This workflow resulted in a yield of approximately 10 mg enamelin per liter culture. Optimal conditions for IMAC purification were obtained using Ni2+ as the metal ion, and when including 30 mM imidazole during binding and washing steps. Furthermore, in vitro mineralization assays demonstrated that the recombinant enamelin could promote calcium phosphate mineralization at a concentration of 0.5 mg/ml. CONCLUSIONS: These findings address the scarcity of enamelin by facilitating its accessibility for further investigations into the mechanism of enamel formation and open new avenues for developing enamel-inspired mineralized biomaterials.

Effects of calcium phosphate and phosphorus-dissolving bacteria on microbial structure and function during Torreya Grandis branch waste composting.

BACKGROUND BURKHOLDERIA: is a phosphorus solubilizing microorganism discovered in recent years, which can dissolve insoluble phosphorus compounds into soluble phosphorus. To investigate the effects of Burkholderia and calcium phosphate on the composting of Torreya grandis branches and leaves, as well as to explain the nutritional and metabolic markers related to the composting process. METHODS: In this study, we employed amplicon sequencing and untargeted metabolomics analysis to examine the interplay among phosphorus (P) components, microbial communities, and metabolites during T. grandis branch and leaf waste composting that underwent treatment with calcium phosphate and phosphate-solubilizing bacteria (Burkholderia). There were four composting treatments, 10% calcium phosphate (CaP) or 5 ml/kg (1 × 108/ml Burkholderia) microbial inoculum (WJP) or both (CaP + WJP), and the control group (CK). RESULTS: The results indicated that Burkholderia inoculation and calcium phosphate treatment affected the phosphorus composition, pH, EC, and nitrogen content. Furthermore, these treatments significantly affected the diversity and structure of bacterial and fungal communities, altering microbial and metabolite interactions. The differential metabolites associated with lipids and organic acids and derivatives treated with calcium phosphate treatment are twice as high as those treated with Burkholderia in both 21d and 42d. The results suggest that calcium phosphate treatment alters the formation of some biological macromolecules. CONCLUSION: Both Burkholderia inoculation and calcium phosphate treatment affected the phosphorus composition, nitrogen content and metabolites of T. grandis branch and leaf waste compost.These results extend our comprehension of the coupling of matter transformation and community succession in composting with the addition of calcium phosphate and phosphate-solubilizing bacteria.

Genomic insight of phosphate solubilization and plant growth promotion of two taxonomically distinct winter crops by Enterobacter sp. DRP3.

AIMS: Study of rhizospheric microbiome-mediated plant growth promotional attributes currently highlighted as a key tool for the development of suitable bio-inoculants for sustainable agriculture purposes. In this context, we have conducted a detailed study regarding the characterization of phosphate solubilizing potential by plant growth-promoting bacteria that have been isolated from the rhizosphere of a pteridophyte Dicranopteris sp., growing on the lateritic belt of West Bengal. METHODS AND RESULTS: We have isolated three potent bacterial strains, namely DRP1, DRP2, and DRP3 from the rhizoids-region of Dicranopteris sp. Among the isolated strains, DRP3 is found to have the highest phosphate solubilizing potentiality and is able to produce 655.89 and 627.58 µg ml-1 soluble phosphate by solubilizing tricalcium phosphate (TCP) and Jordan rock phosphate, respectively. This strain is also able to solubilize Purulia rock phosphate moderately (133.51 µg ml-1). Whole-genome sequencing and further analysis of the studied strain revealed the presence of pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase gdh gene along with several others that were well known for their role in phosphate solubilization. Further downstream, quantitative reverse transcriptase PCR-based expression study revealed 1.59-fold upregulation of PQQ-dependent gdh gene during the solubilization of TCP. Root colonization potential of the studied strain on two taxonomically distinct winter crops viz. Cicer arietinum and Triticum aestivum has been checked by using scanning electron microscopy. Other biochemical analyses for plant growth promotion traits including indole acetic acid production (132.02 µg ml-1), potassium solubilization (3 mg l-1), biofilm formation, and exopolymeric substances productions (1.88-2.03 µg ml-1) also has been performed. CONCLUSION: This study highlighted the active involvement of PQQ-dependent gdh gene during phosphate solubilization from any Enterobacter group. Moreover, our study explored different roadmaps for sustainable farming methods and the preservation of food security without endangering soil health in the future.

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.

Genomic insights into organic acid production and plant growth promotion by different species of phosphate-solubilizing bacteria.

Bacteria can solubilize phosphorus (P) through the secretion of low-molecular-weight organic acids and acidification. However, the genes involved in the production of these organic acids are poorly understood. The objectives of this study were to verify the calcium phosphate solubilization and the production of low-molecular-weight organic acids by diverse genera of phosphate solubilizing bacterial strains (PSBS); to identify the genes related to the synthesis of the organic acids in the genomes of these strains and; to evaluate growth and nutrient accumulation of maize plants inoculated with PSBS and fertilized with Bayóvar rock phosphate. Genomic DNA was extracted for strain identification and annotation of genes related to the organic acids production. A greenhouse experiment was performed with five strains plus 150 mg dm- 3 P2O5 as Bayóvar rock phosphate (BRP) to assess phosphate solubilization contribution to maize growth and nutrition. Paraburkholderia fungorum UFLA 04-21 and Pseudomonas anuradhapurensis UFPI B5-8A solubilized over 60% of Ca phosphate and produced high amounts of citric/maleic and gluconic acids in vitro, respectively. Eleven organic acids were identified in total, although not all strains produced all acids. Besides, enzymes related to the organic acids production were found in all bacterial genomes. Plants inoculated with strains UFPI B5-6 (Enterobacter bugandensis), UFPI B5-8A, and UFLA 03-10 (Paenibacillus peoriae) accumulated more biomass than the plants fertilized with BRP only. Strains UFLA 03-10 and UFPI B5-8A increased the accumulation of most macronutrients, including P. Collectively, the results show that PSBS can increase maize growth and nutrient accumulation based on Bayóvar rock phosphate fertilization.

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.

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.

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.

Enzymatic mineralization generates ultrastiff and tough hydrogels with tunable mechanics.

The cartilage and skin of animals, which are made up of more than fifty per cent water, are rather stiff (having elastic moduli of up to 100 megapascals) as well as tough and hard to break (with fracture energies of up to 9,000 joules per square metre). Such features make these biological materials mechanically superior to existing synthetic hydrogels. Lately, progress has been made in synthesizing tough hydrogels, with double-network hydrogels achieving the toughness of skin and inorganic-organic composites showing even better performance. However, these materials owe their toughness to high stretchability; in terms of stiffness, synthetic hydrogels cannot compete with their natural counterparts, with the best examples having elastic moduli of just 10 megapascals or less. Previously, we described the enzyme-induced precipitation and crystallization of hydrogels containing calcium carbonate, but the resulting materials were brittle. Here we report the enzyme-induced formation of amorphous calcium phosphate nanostructures that are homogenously distributed within polymer hydrogels. Our best materials have fracture energies of 1,300 joules per square metre even in their fully water-swollen state-a value superior to that of most known water-swollen synthetic materials. We are also able to modulate their stiffness up to 440 megapascals, well beyond that of cartilage and skin. Furthermore, the highly filled composite materials can be designed to be optically transparent and to retain most of their stretchability even when notched. We show that percolation drives the mechanical properties, particularly the high stiffness, of our uniformly mineralized hydrogels.

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.

Octacalcium Phosphate/Gelatin Composite (OCP/Gel) Enhances Bone Repair in a Critical-sized Transcortical Femoral Defect Rat Model.

BACKGROUND: Bone grafting is widely used to treat large bone defects. A porous composite of a bioactive octacalcium phosphate material with gelatin sponge (OCP/Gel) has been shown to biodegrade promptly and be replaced with new bone both in animal models of a membranous bone defect and a long bone defect. However, it is unclear whether OCP/Gel can regenerate bone in more severe bone defects, such as a critical-size transcortical defect. QUESTIONS/PURPOSES: Using an in vivo rat femur model of a standardized, transcortical, critical-size bone defect, we asked: Compared with a Gel control, does OCP/Gel result in more newly formed bone as determined by (1) micro-CT evaluation, (2) histologic and histomorphometric measures, and (3) osteocalcin staining and tartrate-resistant acid phosphatase staining? METHODS: Thirty-four 12-week-old male Sprague-Dawley rats (weight 356 ± 25.6 g) were used. Gel and OCP/Gel composites were prepared in our laboratory. Porous cylinders 3 mm in diameter and 4 mm in height were manufactured from both materials. The OCP/Gel and Gel cylinders were implanted into a 3-mm-diameter transcortical critical-size bone defect model in the left rat femur. The OCP/Gel and Gel were randomly assigned, and the cylinders were implanted. The biological responses of the defect regions were evaluated radiologically and histologically. At 4 and 8 weeks after implantation, CT evaluation, histological examination of decalcified samples, and immunostaining were quantitatively performed to evaluate new bone formation and remaining bone graft substitutes and activity of osteoblasts and osteoclast-like cells (n = 24). Qualitative histological evaluation was performed on undecalcified samples at 3 weeks postimplantation (n = 10). CT and decalcified tissue analysis was not performed blinded, but an analysis of undecalcified specimens was performed under blinded conditions. RESULTS: Radiologic analysis revealed that the OCP/Gel group showed radiopaque regions around the OCP granules and at the edge of the defect margin 4 weeks after implantation, suggesting that new bone formation occurred in two ways. In contrast, the rat femurs in the Gel group had a limited radiopaque zone at the edge of the defect region. The amount of new bone volume analyzed by micro-CT was higher in the OCP/Gel group than in the Gel group at 4 and 8 weeks after implantation (​​4 weeks after implantation: OCP/Gel versus Gel: 6.1 ± 1.6 mm 3 versus 3.4 ± 0.7 mm 3 , mean difference 2.7 [95% confidence interval (CI) 0.9 to 4.5]; p = 0.002; intraclass correlation coefficient [ICC] 0.72 [95% CI 0.29 to 0.91]; 8 weeks after implantation: OCP/Gel versus Gel: 3.9 ± 0.7 mm 3 versus 1.4 ± 1.1 mm 3 , mean difference 2.5 [95% CI 0.8 to 4.3]; p = 0.004; ICC 0.81 [95% CI 0.47 to 0.94]). Histologic evaluation also showed there was a higher percentage of new bone formation in the OCP/Gel group at 4 and 8 weeks after implantation (​​4 weeks after implantation: OCP/Gel versus Gel: 31.2% ± 5.3% versus 13.6% ± 4.0%, mean difference 17.6% [95% CI 14.2% to 29.2%]; p < 0.001; ICC 0.83 [95% CI 0.53 to 0.95]; 8 weeks after implantation: OCP/Gel versus Gel: 28.3% ± 6.2% versus 9.5% ± 1.9%, mean difference 18.8% [95% CI 11.3% to 26.3%]; p < 0.001; ICC 0.90 [95% CI 0.69 to 0.97]). Bridging of the defect area started earlier in the OCP/Gel group than in the Gel group at 4 weeks after implantation. Osteocalcin immunostaining showed that the number of mature osteoblasts was higher in the OCP/Gel group than in the Gel group at 4 weeks (OCP/Gel versus Gel: 42.1 ± 6.5/mm 2 versus 17.4 ± 5.4/mm 2 , mean difference 24.7 [95% CI 16.2 to 33.2]; p < 0.001; ICC 0.99 [95% CI 0.97 to 0.99]). At 4 weeks, the number of osteoclast-like cells was higher in the OCP/Gel composite group than in the Gel group (OCP/Gel versus Gel: 3.2 ± 0.6/mm 2 versus 0.9 ± 0.4/mm 2 , mean difference 2.3 [95% CI 1.3 to 3.5]; p < 0.001; ICC 0.79 [95% CI 0.35 to 0.94]). CONCLUSION: OCP/Gel composites induced early bone remodeling and cortical bone repair in less time than did the Gel control in a rat critical-size, transcortical femoral defect, suggesting that OCP/Gel could be used as a bone replacement material to treat severe bone defects. CLINICAL RELEVANCE: In a transcortical bone defect model of critical size in the rat femur, the OCP/Gel composite demonstrated successful bone regeneration. Several future studies are needed to evaluate the clinical application of this interesting bone graft substitute, including bone formation capacity in refractory fracture and spinal fusion models and the comparison of bone strength after repair with OCP/Gel composite to that of autologous bone.

A diecast mineralization process forms the tough mantis shrimp dactyl club.

Biomineralization, the process by which mineralized tissues grow and harden via biogenic mineral deposition, is a relatively lengthy process in many mineral-producing organisms, resulting in challenges to study the growth and biomineralization of complex hard mineralized tissues. Arthropods are ideal model organisms to study biomineralization because they regularly molt their exoskeletons and grow new ones in a relatively fast timescale, providing opportunities to track mineralization of entire tissues. Here, we monitored the biomineralization of the mantis shrimp dactyl club-a model bioapatite-based mineralized structure with exceptional mechanical properties-immediately after ecdysis until the formation of the fully functional club and unveil an unusual development mechanism. A flexible membrane initially folded within the club cavity expands to form the new club's envelope. Mineralization proceeds inwards by mineral deposition from this membrane, which contains proteins regulating mineralization. Building a transcriptome of the club tissue and probing it with proteomic data, we identified and sequenced Club Mineralization Protein 1 (CMP-1), an abundant mildly phosphorylated protein from the flexible membrane suggested to be involved in calcium phosphate mineralization of the club, as indicated by in vitro studies using recombinant CMP-1. This work provides a comprehensive picture of the development of a complex hard tissue, from the secretion of its organic macromolecular template to the formation of the fully functional club.

Amelogenesis: Transformation of a protein-mineral matrix into tooth enamel.

During enamel formation, the organic enamel protein matrix interacts with calcium phosphate minerals to form elongated, parallel, and bundled enamel apatite crystals of extraordinary hardness and biomechanical resilience. The enamel protein matrix consists of unique enamel proteins such as amelogenin, ameloblastin, and enamelin, which are secreted by highly specialized cells called ameloblasts. The ameloblasts also facilitate calcium and phosphate ion transport toward the enamel layer. Within ameloblasts, enamel proteins are transported as a polygonal matrix with 5 nm subunits in secretory vesicles. Upon expulsion from the ameloblasts, the enamel protein matrix is re-organized into 20 nm subunit compartments. Enamel matrix subunit compartment assembly and expansion coincide with C-terminal cleavage by the MMP20 enamel protease and N-terminal amelogenin self-assembly. Upon enamel crystal precipitation, the enamel protein phase is reconfigured to surround the elongating enamel crystals and facilitate their elongation in C-axis direction. At this stage of development, and upon further amelogenin cleavage, central and polyproline-rich fragments of the amelogenin molecule associate with the growing mineral crystals through a process termed "shedding", while hexagonal apatite crystals fuse in longitudinal direction. Enamel protein sheath-coated enamel "dahlite" crystals continue to elongate until a dense bundle of parallel apatite crystals is formed, while the enamel matrix is continuously degraded by proteolytic enzymes. Together, these insights portrait enamel mineral nucleation and growth as a complex and dynamic set of interactions between enamel proteins and mineral ions that facilitate regularly seeded apatite growth and parallel enamel crystal elongation.

Structure and mineralization of the spearing mantis shrimp (Stomatopoda; Lysiosquillina maculata) body and spike cuticles.

Stomatopoda is a crustacean order including sophisticated predators called spearing and smashing mantis shrimps that are separated from the well-studied Eumalacotraca since the Devonian. The spearing mantis shrimp has developed a spiky dactyl capable of impaling fishes or crustaceans in a fraction of second. In this high velocity hunting technique, the spikes undergo an intense mechanical constraint to which their exoskeleton (or cuticle) has to be adapted. To better understand the spike cuticle internal architecture and composition, electron microscopy, X-ray microanalysis and Raman spectroscopy were used on the spikes of 7 individuals (collected in French Polynesia and Indonesia), but also on parts of the body cuticle that have less mechanical stress to bear. In the body cuticle, several specificities linked to the group were found, allowing to determine the basic structure from which the spike cuticle has evolved. Results also highlighted that the body cuticle of mantis shrimps could be a model close to the ancestral arthropod cuticle by the aspect of its biological layers (epi- and procuticle including exo- and endocuticle) as well as by the Ca-carbonate/phosphate mineral content of these layers. In contrast, the spike cuticle exhibits a deeply modified organization in four functional regions overprinted on the biological layers. Each of them has specific fibre arrangement or mineral content (fluorapatite, ACP or phosphate-rich Ca-carbonate) and is thought to assume specific mechanical roles, conferring appropriate properties on the entire spike. These results agree with an evolution of smashing mantis shrimps from primitive stabbing/spearing shrimps, and thus also allowed a better understanding of the structural modifications described in previous studies on the dactyl club of smashing mantis shrimps.

Amelogenin phosphorylation regulates tooth enamel formation by stabilizing a transient amorphous mineral precursor.

Dental enamel comprises interwoven arrays of extremely long and narrow crystals of carbonated hydroxyapatite called enamel rods. Amelogenin (AMELX) is the predominant extracellular enamel matrix protein and plays an essential role in enamel formation (amelogenesis). Previously, we have demonstrated that full-length AMELX forms higher-order supramolecular assemblies that regulate ordered mineralization in vitro, as observed in enamel rods. Phosphorylation of the sole AMELX phosphorylation site (Ser-16) in vitro greatly enhances its capacity to stabilize amorphous calcium phosphate (ACP), the first mineral phase formed in developing enamel, and prevents apatitic crystal formation. To test our hypothesis that AMELX phosphorylation is critical for amelogenesis, we generated and characterized a hemizygous knockin (KI) mouse model with a phosphorylation-defective Ser-16 to Ala-16 substitution in AMELX. Using EM analysis, we demonstrate that in the absence of phosphorylated AMELX, KI enamel lacks enamel rods, the hallmark component of mammalian enamel, and, unlike WT enamel, appears to be composed of less organized arrays of shorter crystals oriented normal to the dentinoenamel junction. KI enamel also exhibited hypoplasia and numerous surface defects, whereas heterozygous enamel displayed highly variable mosaic structures with both KI and WT features. Importantly, ACP-to-apatitic crystal transformation occurred significantly faster in KI enamel. Secretory KI ameloblasts also lacked Tomes' processes, consistent with the absence of enamel rods, and underwent progressive cell pathology throughout enamel development. In conclusion, AMELX phosphorylation plays critical mechanistic roles in regulating ACP-phase transformation and enamel crystal growth, and in maintaining ameloblast integrity and function during amelogenesis.

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 ß-glycerophosphate (ßG), 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 ßG (by day 11). A redshift of the phosphate stretching peak for cells supplemented with ßG 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.