The Na+/Ca2+ exchanger NCX3 mediates Ca2+ entry into matrix vesicles to facilitate initial steps of mineralization in osteoblasts.

Matrix vesicles (MVs) provide the initial site for amorphous hydroxyapatite (HA) formation within mineralizing osteoblasts. Although Na+/Ca2+ exchanger isoform-3 (NCX3, SLC8A3) was presumed to function as major Ca2+ transporter responsible for Ca2+ extrusion out of osteoblast into the calcifying bone matrix, its presence and functional role in MVs have not been investigated. In this study, we investigated the involvement of NCX3 in MV-mediated mineralization process and its impact on bone formation. Using differentiated MC3T3-E1 cells, we demonstrated that NCX3 knockout in these cells resulted in a significant reduction of Ca2+ deposition due to reduced Ca2+ entry within the MVs, leading to impaired mineralization. Consequently, the capacity of MVs to promote extracellular HA formation was diminished. Moreover, primary osteoblast isolated from NCX3 deficient mice (NCX3-/-) exhibits reduced mineralization efficacy without any effect on osteoclast activity. To validate this in vitro finding, µCT analysis revealed a substantial decrease in trabecular bone mineral density in both genders of NCX3-/- mice, thus supporting the critical role of NCX3 in facilitating Ca2+ uptake into the MVs to initiate osteoblast-mediated mineralization. NCX3 expression was also found to be the target of downregulation by inflammatory mediators in vitro and in vivo. This newfound understanding of NCX3's functional role in MVs opens new avenues for therapeutic interventions aimed at enhancing bone mineralization and treating mineralization-related disorders.

Rubble persistence under ocean acidification threatened by accelerated bioerosion and lower-density coral skeletons.

As the balance between erosional and constructive processes on coral reefs tilts in favor of framework loss under human-induced local and global change, many reef habitats worldwide degrade and flatten. The resultant generation of coral rubble and the beds they form can have lasting effects on reef communities and structural complexity, threatening the continuity of reef ecological functions and the services they provide. To comprehensively capture changing framework processes and predict their evolution in the context of climate change, heavily colonized rubble fragments were exposed to ocean acidification (OA) conditions for 55 days. Controlled diurnal pH oscillations were incorporated in the treatments to account for the known impact of diel carbonate chemistry fluctuations on calcification and dissolution response to OA. Scenarios included contemporary pH (8.05 ± 0.025 diel fluctuation), elevated OA (7.90 ± 0.025), and high OA (7.70 ± 0.025). We used a multifaceted approach, combining chemical flux analyses, mass alteration measurements, and computed tomography scanning images to measure total and chemical bioerosion, as well as chemically driven secondary calcification. Rates of net carbonate loss measured in the contemporary conditions (1.36 kg m-2 year-1) were high compared to literature and increased in OA scenarios (elevated: 1.84 kg m-2 year-1 and high: 1.59 kg m-2 year-1). The acceleration of these rates was driven by enhanced chemical dissolution and reduced secondary calcification. Further analysis revealed that the extent of these changes was contingent on the density of the coral skeleton, in which the micro- and macroborer communities reside. Findings indicated that increased mechanical bioerosion rates occurred in rubble with lower skeletal density, which is of note considering that corals form lower-density skeletons under OA. These direct and indirect effects of OA on chemical and mechanical framework-altering processes will influence the permanence of this crucial habitat, carrying implications for biodiversity and reef ecosystem function.

Correlative geochemical imaging of Desmophyllum dianthus reveals biomineralisation strategy as a key coral vital effect.

The chemical and isotopic composition of stony coral skeletons form an important archive of past climate. However, these reconstructions are largely based on empirical relationships often complicated by "vital effects" arising from uncertain physiological processes of the coral holobiont. The skeletons of deep-sea corals, such as Desmophyllum dianthus, are characterised by micron-scale or larger geochemical heterogeneity associated with: (1) centres of calcification (COCs) where nucleation of new skeleton begins, and (2) fibres that thicken the skeleton. These features are difficult to sample cleanly using traditional techniques, resulting in uncertainty surrounding both the causes of geochemical differences and their influence on environmental signals. Here we combine optical, and in-situ chemical and isotopic, imaging tools across a range of spatial resolutions (~ 100 nm to 10 s of µm) in a correlative multimodal imaging (CMI) approach to isolate the microstructural geochemistry of each component. This reveals COCs are characterised by higher organic content, Mg, Li and Sr and lower U, B and δ11B compared to fibres, reflecting the contrasting biomineralisation mechanisms employed to construct each feature. CMI is rarely applied in Environmental/Earth Sciences, but here we illustrate the power of this approach to unpick the "vital effects" in D. dianthus, and by extension, other scleractinian corals.

17ß-Estradiol (E2) Activates Matrix Mineralization through Genomic/Nongenomic Pathways in MC3T3-E1 Cells.

Estrogen plays an important role in osteoporosis prevention. We herein report the possible novel signaling pathway of 17ß-estradiol (E2) in the matrix mineralization of MC3T3-E1, an osteoblast-like cell line. In the culture media-containing stripped serum, in which small lipophilic molecules such as steroid hormones including E2 were depleted, matrix mineralization was significantly reduced. However, the E2 treatment induced this. The E2 effects were suppressed by ICI182,780, the estrogen receptor (ER)α, and the ERß antagonist, as well as their mRNA knockdown, whereas Raloxifene, an inhibitor of estrogen-induced transcription, and G15, a G-protein-coupled estrogen receptor (GPER) 1 inhibitor, had little or no effect. Furthermore, the E2-activated matrix mineralization was disrupted by PMA, a PKC activator, and SB202190, a p38 MAPK inhibitor, but not by wortmannin, a PI3K inhibitor. Matrix mineralization was also induced by the culture media from the E2-stimulated cell culture. This effect was hindered by PMA or heat treatment, but not by SB202190. These results indicate that E2 activates the p38 MAPK pathway via ERs independently from actions in the nucleus. Such activation may cause the secretion of certain signaling molecule(s), which inhibit the PKC pathway. Our study provides a novel pathway of E2 action that could be a therapeutic target to activate matrix mineralization under various diseases, including osteoporosis.

OPN, BSP, and Bone Quality-Structural, Biochemical, and Biomechanical Assessment in OPN-/-, BSP-/-, and DKO Mice.

Osteopontin (OPN) and Bone Sialoprotein (BSP), abundantly expressed by osteoblasts and osteoclasts, appear to have important, partly overlapping functions in bone. In gene-knockout (KO, -/-) models of either protein and their double (D)KO in the same CD1/129sv genetic background, we analyzed the morphology, matrix characteristics, and biomechanical properties of femur bone in 2 and 4 month old, male and female mice. OPN-/- mice display inconsistent, perhaps localized hypermineralization, while the BSP-/- are hypomineralized throughout ages and sexes, and the low mineralization of young DKO mice recovers with age. The higher contribution of primary bone remnants in OPN-/- shafts suggests a slow turnover, while their lower percentage in BSP-/- indicates rapid remodeling, despite FTIR-based evidence in this genotype of a high maturity of the mineralized matrix. In 3-point bending assays, OPN-/- bones consistently display higher Maximal Load, Work to Max. Load and in young mice Ultimate Stress, an intrinsic characteristic of the matrix. Young male and old female BSP-/- also display high Work to Max. Load along with low Ultimate Stress. Principal Component Analysis confirms the major role of morphological traits in mechanical competence, and evidences a grouping of the WT phenotype with the OPN-/- and of BSP-/- with DKO, driven by both structural and matrix parameters, suggesting that the presence or absence of BSP has the most profound effects on skeletal properties. Single or double gene KO of OPN and BSP thus have multiple distinct effects on skeletal phenotypes, confirming their importance in bone biology and their interplay in its regulation.

Harnessing oriented arrangement of collagen fibers by 3D printing for enhancing mechanical and osteogenic properties of mineralized collagen scaffolds.

As the structural basis of connective and load-bearing tissues, collagen fibers with orientation play an important role in the mechanical properties and physiological and biochemical functions of the tissues, but viable methods for preparing scaffolds with highly oriented collagenous structure still need to be further studied. In this study, pure collagen was used as printing ink to 3D printing. Harnessing oriented collagen fiber structure by 3D printing for promoting mechanical and osteogenic properties of scaffolds. The scaffolds with different printed angles and thicknesses were prepared to fit the bone defect site and realize personalized customization. The orientation assembly of collagen fibers was promoted by shear force action of 3D printing, the regular arrangement of collagen fibers and stabilization of fiber structure were promoted by pH adjustment and glutaraldehyde cross-linking, and the collagen fibers were mineralized by cyclic mineralization method. The microscopic morphology of fiber arrangement in the scaffolds were investigated by scanning electron microscopy. Results demonstrated that collagen fibers were changed from non-oriented to oriented after 3D printing. And the tensile modulus of the scaffolds with oriented collagen fibers was nine times higher than that of the scaffolds with non-oriented fibers. Moreover, the effects of oriented collagen fibers on the proliferation, differentiation and mineralization of MC3T3-E1 cells were studied by CCK-8 assay, live/dead cell staining, alkaline phosphatase activity test, and Alizarin red staining. The results indicated that cell proliferation, differentiation and mineralization were significantly promoted by oriented collagen fibers, and the cells proliferated directionally in the direction of the fibers. Taken together, mineralized collagen fiber scaffolds with oriented collagen fibers have great potential in bone tissue engineering applications.

Controlled delivery of procyanidin through magnesium oxide nanoparticles (MgO NPs) to improve the activity and mineralization of osteoblasts under oxidative stressin vitro.

Excessive reactive oxygen species (ROS) in the microenvironment of osteoporosis (OP) not only accelerate the bone absorption, but also affect the osteogenic and mineralized effect of osteoblasts. Procyanidins (PC) have been reported to have anti-oxidation effects, but low bioavailability. This study aimed to explore the effect of magnesium oxide nanoparticles (MgO-PC NPs)-loaded PC on the osteogenesis and mineralization of osteoblasts that stimulated by H2O2. PC was loaded onto MgO NPs and characterized by transmission electron microscopy, energy dispersive spectroscopy, dynamic light scattering, and Fourier transform infrared spectroscopy. After primary screening by cytotoxicity assay, MgO-PC NPs containing 20 µM of PC were chosen for further studies. In H2O2-stimulated osteoblasts, dichlorodihydrofluorescein diacetate probe, Cell Counting Kit-8, quantitative real-time polymerase chain reaction, alkaline phosphatase staining/activity and Alizarin red staining were used to detect the ROS production, cell viability and osteogenic and mineralized markers of osteoblasts. PC was loaded onto MgO NPs to successfully receive MgO-PC NPs with a diameter of about 144 nm and negative potential. PC can sustain release from MgO-PC NPs for at least 16 d. The controlled release of PC from MgO-PC NPs can effectively eliminate ROS and thereby promoted the cell activity. Most importantly, the osteogenesis and mineralization of osteoblasts under oxidative stress were also significantly reversed by MgO-PC NPS. Thus, these findings indicate that MgO-PC NPs may be developed as a potential therapeutic strategy for OP.

Effects of heterogeneous surface characteristics on hemocompatibility and cytocompatibility of bacterial nanocellulose.

The surface properties of cardiovascular biomaterials play a critical role in their biological responses. Although bacterial nanocellulose (BNC) materials have exhibited potential applications in cardiovascular implants, the impact of their surface characteristics on biocompatibility has rarely been studied. This study investigated the mechanism for the biocompatibility induced by the physicochemical properties of both sides of BNC. With greater wettability and smoothness, the upper BNC surface reduced protein adsorption by 25 % compared with the lower surface. This prolonged the plasma re-calcification time by 14 % in venous blood. Further, compared with the lower BNC surface, the upper BNC surface prolonged the activated partial thromboplastin time by 5 % and 4 % in arterial and venous blood, respectively. Moreover, the lower BNC surface with lesser rigidity, higher roughness, and sparser fiber structure promoted cell adhesion. The lower BNC surface enhanced the proliferation rate of L929 and HUVECs cells by 15 % and 13 %, respectively, compared with the upper BNC surface. With lesser stiffness, the lower BNC surface upregulated the expressions of CD31 and eNOS while down-regulating the ICAM-1 expression - This promoted the proliferation of HUVECs. The findings of this study will provide valuable insights into the design of blood contact materials and cardiovascular implants.

Dynamics of bone mineralization in primiparous sows as a function of dietary phosphorus and calcium during lactation.

To maximize the efficiency of dietary P utilization in swine production, understanding the mechanisms of P utilization in lactating sows is relevant due to their high P requirement and the resulting high inorganic P intake. Gaining a better knowledge of the Ca and P quantities that can be mobilized from bones during lactation, and subsequently replenished during the following gestation, would enable the development of more accurate P requirements incorporating this process of bone dynamics. The objective was to measure the amount of body mineral reserves mobilized during lactation, depending on dietary digestible P and phytase addition and to measure the amount recovered during the following gestation. Body composition of 24 primiparous sows was measured by dual-energy x-ray absorptiometry 2, 14, 26, 70 and 110 days after farrowing. Four lactation diets were formulated to cover nutritional requirements, with the exception of Ca and digestible P: 100% (Lact100; 9.9 g Ca and 3.0 g digestible P/kg), 75% (Lact75), 50% without added phytase (Lact50) and 50% with added phytase (Lact50 + FTU). The gestation diet was formulated to cover the nutritional requirements of Ca and digestible P (8.2 g Ca and 2.6 g digestible P/kg). During the 26 days of lactation, each sow mobilized body mineral reserves. The mean amount of mobilized bone mineral content (BMC) was 664 g, representing 240 g Ca and 113 g P. At weaning, the BMC (g/kg of BW) of Lact50 sows tended to be lower than Lact100 sows (-12.8%, linear Ca and P effect × quadratic time effect) while the BMC of Lact50 + FTU sows remained similar to that of Lact100 sows. During the following gestation, BMC returned to similar values among treatments. Therefore, the sows fed Lact50 could recover from the higher bone mineral mobilization that occurred during lactation. The P excretion was reduced by 40 and 43% in sows fed Lact50 and Lact50 + FTU, respectively, relative to sows fed Lact100. In conclusion, the quantified changes in body composition during the lactation and following gestation of primiparous sows show that bone mineral reserves were mobilized and recovered and that its degree was dependent on the dietary P content and from phytase supplementation during lactation. In the future, considering this potential of the sows' bone mineralization dynamics within the factorial assessment of P requirement and considering the digestible P equivalency of microbial phytase could greatly limit the dietary use of inorganic phosphates and, thus, reduce P excretion.

Effects of LP533401 on vascular and bone calcification in hyperlipidemic mice.

Peripheral serotonin levels are associated with cardiovascular disease risk. We previously found that serum serotonin levels are higher in hyperlipidemic mice than wild-type mice. Evidence also suggests that serotonin regulates biomineralization, in that serotonin treatment augments TNF-a-induced matrix calcification of aortic valve interstitial cells and that a selective inhibitor of peripheral serotonin, LP533401, rescues bone loss induced by ovariectomy in mice. Thus, in the present study, we examined the effects of LP533401 on both skeletal bone mineral density (BMD) and aortic calcification in both young and older hyperlipidemic mice susceptible to calcific atherosclerosis and bone loss. By serial in vivo microCT imaging, we assessed BMD and aortic calcification of Apoe-/- mice fed an atherogenic (high cholesterol) diet alone or mixed with LP533401. Results show that in the young mice, LP533401 blunted skeletal bone loss in lumbar vertebrae but not in femurs. LP533401 also blunted the initial development of aortic calcification but not its progression. Echocardiographic analysis showed that LP533401 blunted both hyperlipidemia-induced cardiac hypertrophy and left ventricular dysfunction. In the older mice, LP533401 increased the BMD of lumbar vertebrae but not of femurs. The aortic calcification progressed in both controls and LP533401-treated mice, but, at post-treatment, LP533401-treated mice had significantly less aortic calcification than the controls. These findings suggest that LP533401 mitigates adverse effects of hyperlipidemia on skeletal and vascular tissues in site- and stage-dependent manners.

Diagnostic survey of analytical methods used to determine bone mineralization in pigs.

Pigs from 64 commercial sites across 14 production systems in the Midwest United States were evaluated for baseline biological measurements used to determine bone mineralization. There were three pigs selected from each commercial site representing: 1) a clinically normal pig (healthy), 2) a pig with evidence of clinical lameness (lame), and 3) a pig from a hospital pen that was assumed to have recent low feed intake (unhealthy). Pigs ranged in age from nursery to market weight, with the three pigs sampled from each site representing the same age or phase of production. Blood, urine, metacarpal, fibula, 2nd rib, and 10th rib were collected and analyzed. Each bone was measured for density and ash (defatted and non-defatted technique). A bone × pig type interaction (P < 0.001) was observed for defatted and non-defatted bone ash and density. For defatted bone ash, there were no differences among pig types for the fibulas, 2nd rib, and 10th rib (P > 0.10), but metacarpals from healthy pigs had greater (P < 0.05) percentage bone ash compared to unhealthy pigs, with the lame pigs intermediate. For non-defatted bone ash, there were no differences among pig types for metacarpals and fibulas (P > 0.10), but unhealthy pigs had greater (P < 0.05) non-defatted percentage bone ash for 2nd and 10th ribs compared to healthy pigs, with lame pigs intermediate. Healthy and lame pigs had greater (P < 0.05) bone density than unhealthy pigs for metacarpals and fibulas, with no difference observed for ribs (P > 0.10). Healthy pigs had greater (P < 0.05) serum Ca and 25(OH)D3 compared to unhealthy pigs, with lame pigs intermediate. Healthy pigs had greater (P < 0.05) serum P compared to unhealthy and lame pigs, with no differences between the unhealthy and lame pigs. Unhealthy pigs excreted significantly more (P < 0.05) P and creatinine in the urine compared to healthy pigs with lame pigs intermediate. In summary, there are differences in serum Ca, P, and vitamin D among healthy, lame, and unhealthy pigs. Differences in bone mineralization among pig types varied depending on the analytical procedure and bone, with a considerable range in values within pig type across the 14 production systems sampled.

There is little literature or data comparing bone diagnostic results for healthy, lame, and unhealthy pigs. Typically, diagnosticians assessing clinical lameness cases in pigs will measure bone mineralization along with histopathological evaluation to diagnose and assess the severity of metabolic bone disease. Bone ash is the primary method to determine bone mineralization, with the removal of the lipid in the bone (defatting) before the bone is ashed, compared to not removing the lipid before the ashing (non-defatted). Defatting the bone reduces the amount of variation across the bones compared to non-defatting. In this diagnostic survey, there was no difference among the healthy, lame, or unhealthy pigs when comparing defatted bone ash, however, unhealthy pigs had an increased bone ash percentage compared to the healthy and lame pigs when the bones were assessed using the non-defatted procedure. There was variation across production systems and pig types for serum vitamin D. When comparing the pig types, healthy pigs had increased serum Ca, P, and vitamin D [25(OH)D3] compared to the unhealthy pigs, with the lame pigs intermediate.

Laser-assisted surface alloying of titanium with silver to enhance antibacterial and bone-cell mineralization properties of orthopedic implants.

Orthopedic device-related infection (ODRI) poses a significant threat to patients with titanium-based implants. The challenge lies in developing antibacterial surfaces that preserve the bulk mechanical properties of titanium implants while exhibiting characteristics similar to bone tissue. In response, we present a two-step approach: silver nanoparticle (AgNP) coating followed by selective laser-assisted surface alloying on commonly used titanium alumina vanadium (TiAl6V4) implant surfaces. This process imparts antibacterial properties without compromising the bulk mechanical characteristics of the titanium alloy. Systematic optimization of laser beam power (8-40 W) resulted in an optimized surface (32 W) with uniform TiAg alloy formation. This surface displayed a distinctive hierarchical mesoporous textured surface, featuring cauliflower-like nanostructures measuring between 5-10 nm uniformly covering spatial line periods of 25 µm while demonstrating homogenous elemental distribution of silver throughout the laser processed surface. The optimized laser processed surface exhibited prolonged superhydrophilicity (40 days) and antibacterial efficacy (12 days) against Staphylococcus aureus and Escherichia coli. Additionally, there was a significant twofold increase in bone mineralization compared to the pristine Ti6Al4V surface (p < 0.05). Rockwell hardness tests confirmed minimal (<1%) change in bulk mechanical properties compared to the pristine surface. This innovative laser-assisted approach, with its precisely tailored surface morphology, holds promise for providing enduring antibacterial and osteointegration properties, rendering it an optimal choice for modifying load-bearing implant devices without altering material bulk characteristics.

Estradiol-17ß and bisphenol A affect growth and mineralization in early life stages of seabass.

Natural and synthetic estrogens are contaminants present in aquatic ecosystems. They can have significant consequences on the estrogen-sensitive functions of organisms, including skeletal development and growth of vertebrate larvae. Synthetic polyphenols represent a group of environmental xenoestrogens capable of binding the receptors for the natural hormone estradiol-17ß (E2). To better understand how (xeno-)estrogens can affect the skeleton in fish species with high ecological and commercial interest, 16 days post-hatch larvae of the seabass were experimentally exposed for 7 days to E2 and Bisphenol A (BPA), both used at the regulatory concentration of surface water quality (E2: 0.4 ng.L-1, BPA: 1.6 µg.L-1) or at a concentration 100 times higher. Skeletal mineralization levels were evaluated using Alizarin red staining, and expression of several genes playing key roles in growth, skeletogenesis and estrogen signaling pathways was assessed by qPCR. Our results show that E2 exerts an overall negative effect on skeletal mineralization at the environmental concentration of 0.4 ng.L-1, correlated with an increase in the expression of genes associated only with osteoblast bone cells. Both BPA exposures inhibited mineralization with less severe effects and modified bone homeostasis by regulating the expression of gene encoding osteoblasts and osteoclasts markers. Our results demonstrate that environmental E2 exposure inhibits larval growth and has an additional inhibitory effect on skeleton mineralization while both BPA exposures have marginal inhibitory effect on skeletal mineralization. All exposures have significant effects on transcriptional levels of genes involved in the skeletal development of seabass larvae.

Calcified Cartilage-Guided Identification of Osteogenic Molecules and Geometries.

Calcified cartilage digested by chondroclasts provides an excellent scaffold to initiate bone formation. We analyzed bioactive proteins and microarchitecture of calcified cartilage either separately or in combination and evaluated biomimetic osteogenic culture conditions of surface-coated micropatterning. To do so, we prepared a crude extract from porcine femoral growth plates, which enhanced in vitro mineralization when coated on flat-bottom culture dishes, and identified four candidate proteins by fractionation and mass spectrometry. Murine homologues of two candidates, desmoglein 4 (DSG4) and peroxiredoxin 6 (PRDX6), significantly promoted osteogenic activity based on in vitro mineralization and osteoblast differentiation. Moreover, we observed DSG4 and PRDX6 protein expression in mouse femur. In addition, we designed circular, triangular, and honeycomb micropatterns with 30 or 50 µm units, either isolated or connected, to mimic hypertrophic chondrocyte-sized compartments. Isolated, larger honeycomb patterns particularly enhanced osteogenesis in vitro. Mineralization on micropatterns was positively correlated with the reduction of osteoblast migration distance in live cell imaging. Finally, we evaluated possible combinatorial effects of coat proteins and micropatterns and observed an additive effect of DSG4 or PRDX6 coating with micropatterns. These data suggest that combining a bioactive surface coating with osteogenic micropatterns may recapitulate initiation of bone formation during endochondral ossification.

Unveiling the role of hydroxyapatite and hydroxyapatite/silver composite in osteoblast-like cell mineralization: An exploration through their viscoelastic properties.

Mechanical properties are becoming fundamental for advancing the comprehension of cellular processes. This study addresses the relationship between viscoelastic properties and the cellular mineralization process. Osteoblast-like cells treated with an osteogenic medium were employed for this purpose. Additionally, the study explores the impact of hydroxyapatite (HA) and hydroxyapatite/silver (HA/Ag) composite on this process. AFM relaxation experiments were conducted to extract viscoelastic parameters using the Fractional Zener (FZ) and Fractional Kelvin (FK) models. Our findings revealed that the main phases of mineralization are associated with alterations in the viscoelastic properties of osteoblast-like cells. Furthermore, HA and HA/Ag treatments significantly influenced changes in the viscoelastic properties of these cells. In particular, the HA/Ag treatment demonstrated a marked enhancement in cell fluidity, suggesting a possible role of silver in accelerating the mineralization process. Moreover, the study underscores the independence observed between fluidity and stiffness, indicating that modifications in one parameter may not necessarily correspond to changes in the other. These findings shed light on the factors involved in the cellular mineralization process and emphasize the importance of using viscoelastic properties to discern the impact of treatments on cells.

Challenging pitfalls in frozen section pathology: a case of mandible ghost cell odontogenic carcinoma and the literature review.

BACKGROUND: Ghost cell odontogenic carcinoma (GCOC) is a rare malignancy characterized by the presence of ghost cells, preferably in the maxilla. Only slightly more than 50 case reports of GCOC have been documented to date. Due to the rarity of this tumor and its nonspecific clinical criteria, there is a heightened risk of misdiagnosis in clinical examination, imaging findings, and pathology interpretation. CASE PRESENTATION: A 50-year-old male patient presented to the hospital due to experiencing pain in his lower front teeth while eating for the past 2 months. Upon examination, a red, hard, painless mass was found in his left lower jaw, measuring approximately 4.0 cm × 3.5 cm. Based on the malignant histological morphology of the tumor and the abundant red-stained keratinized material, the preoperative frozen section pathology misdiagnosed it as squamous cell carcinoma (SCC). The surgical resection specimen pathology via paraffin section revealed that the tumor was characterized by round-like epithelial islands within the fibrous interstitium, accompanied by a large number of ghost cells and some dysplastic dentin with infiltrative growth. The malignant components displayed marked heterogeneity and mitotic activity. Additionally, a calcified cystic tumor component of odontogenic origin was observed. Hemorrhage, necrosis, and calcifications were present, with a foreign body reaction around ghost cells. Immunoreactivity for ß-catenin showed strong nuclear positivity in tumor cells, while immunostaining was completely negative for p53. The Ki67 proliferation index was approximately 30-40%. The tumor cells exhibited diffuse CK5/6, p63, and p40 immunoreactivity, with varying immunopositivity for EMA. Furthermore, no BRAFV600E mutation was identified by ARMS-PCR. The final pathology confirmed that the tumor was a mandible GCOC. CONCLUSION: We have reported and summarized for the first time the specific manifestations of GCOC in frozen section pathology and possible pitfalls in misdiagnosis. We also reviewed and summarized the etiology, pathological features, molecular characteristics, differential diagnosis, imaging features, and current main treatment options for GCOC. Due to its rarity, the diagnosis and treatment of this disease still face certain challenges. A correct understanding of the pathological morphology of GCOC, distinguishing the ghost cells and the secondary stromal reaction around them, is crucial for reducing misdiagnosis rates.

Vertebrate mineralized tissues: A modular structural analysis.

Vertebrate mineralized tissues, present in bones, teeth and scales, have complex 3D hierarchical structures. As more of these tissues are characterized in 3D using mainly FIB SEM at a resolution that reveals the mineralized collagen fibrils and their organization into collagen fibril bundles, highly complex and diverse structures are being revealed. In this perspective we propose an approach to analyzing these tissues based on the presence of modular structures: material textures, pore shapes and sizes, as well as extents of mineralization. This modular approach is complimentary to the widely used hierarchical approach for describing these mineralized tissues. We present a series of case studies that show how some of the same structural modules can be found in different mineralized tissues, including in bone, dentin and scales. The organizations in 3D of the various structural modules in different tissues may differ. This approach facilitates the framing of basic questions such as: are the spatial relations between modular structures the same or similar in different mineralized tissues? Do tissues with similar sets of modules carry out similar functions or can similar functions be carried out using a different set of modular structures? Do mineralized tissues with similar sets of modules have a common developmental or evolutionary pathway? STATEMENT OF SIGNIFICANCE: 3D organization studies of diverse vertebrate mineralized tissues are revealing detailed, but often confusing details about the material textures, the arrangements of pores and differences in the extent of mineralization within a tissue. The widely used hierarchical scheme for describing such organizations does not adequately provide a basis for comparing these tissues, or addressing issues such as structural components thought to be characteristic of bone, being present in dermal tissues and so on. The classification scheme we present is based on identifying structural components within a tissue that can then be systematically compared to other vertebrate mineralized tissues. We anticipate that this classification approach will provide insights into structure-function relations, as well as the evolution of these tissues.

Novel Peptide Derived from Gadus morhua Stimulates Osteoblastic Differentiation and Mineralization through Wnt/ß-Catenin and BMP Signaling Pathways.

Marine biodiversity offers a wide array of active ingredient resources. Gadus morhua peptides (GMPs) showed excellent osteoprotective effects in ovariectomized mice. However, the potential osteogenesis mechanisms of key osteogenic peptides in GMP were seldom reported. In this study, a novel osteogenic peptide (GETNPADSKPGSIR, P-GM-2) was screened from GMP. P-GM-2 has a high stability coefficient and a strong interaction with epidermal growth factor receptor. Cell culture experiments showed that P-GM-2 stimulated the expression of osteogenic differentiation markers to promote osteoblast proliferation, differentiation, and mineralization. Additionally, P-GM-2 phosphorylates GSK-3ß, leading to the stabilization of ß-catenin and its translocation to the nucleus, thus initiating the activation of the Wnt/ß-catenin signaling pathway. Meanwhile, P-GM-2 could also regulate the osteogenic differentiation of preosteoblasts by triggering the BMP/Smad and mitogen-activated protein kinase signaling pathways. Further validation with specific inhibitors (ICG001 and Noggin) demonstrated that the osteogenic activity of P-GM-2 was revealed by the activation of the BMP and Wnt/ß-catenin pathways. In summary, these results provide theoretical and practical insights into P-GM-2 as an effective antiosteoporosis active ingredient.

Using Museum collections to assess the impact of industrialization on mussel (Mytilus edulis) calcification.

Mytilus edulis is a commercially and ecologically important species found along the east coast of the United States. Ecologically, M. edulis improves water quality through filtration feeding and provides habitat formation and coastal protection through reef formation. Like many marine calcifiers, ocean warming, and acidification are a growing threat to these organisms-impacting their morphology and function. Museum collections are useful in assessing long-term environmental impacts on organisms in a natural multi-stressor environment, where acclimation and adaptation can be considered. Using the American Museum of Natural History collections ranging from the early 1900s until now, we show that shell porosity changes through time. Shells collected today are significantly more porous than shells collected in the 1960s and, at some sites, than shells collected from the early 1900s. The disparity between porosity changes matches well with the warming that occurred over the last 130 years in the north Atlantic suggesting that warming is causing porosity changes. However, more work is required to discern local environmental impacts and to fully identify porosity drivers. Since, porosity is known to affect structural integrity, porosity increasing through time could have negative consequences for mussel reef structural integrity and hence habitat formation and storm defenses.

Defining the Most Potent Osteoinductive Culture Conditions for MC3T3-E1 Cells Reveals No Implication of Oxidative Stress or Energy Metabolism.

The MC3T3-E1 preosteoblastic cell line is widely utilised as a reliable in vitro system to assess bone formation. However, the experimental growth conditions for these cells hugely diverge, and, particularly, the osteogenic medium (OSM)'s composition varies in research studies. Therefore, we aimed to define the ideal culture conditions for MC3T3-E1 subclone 4 cells with regard to their mineralization capacity and explore if oxidative stress or the cellular metabolism processes are implicated. Cells were treated with nine different combinations of long-lasting ascorbate (Asc) and ß-glycerophosphate (ßGP), and osteogenesis/calcification was evaluated at three different time-points by qPCR, Western blotting, and bone nodule staining. Key molecules of the oxidative and metabolic pathways were also assessed. It was found that sufficient mineral deposition was achieved only in the 150 µg.mL-1/2 mM Asc/ßGP combination on day 21 in OSM, and this was supported by Runx2, Alpl, Bglap, and Col1a1 expression level increases. NOX2 and SOD2 as well as PGC1α and Tfam were also monitored as indicators of redox and metabolic processes, respectively, where no differences were observed. Elevation in OCN protein levels and ALP activity showed that mineralisation comes as a result of these differences. This work defines the most appropriate culture conditions for MC3T3-E1 cells and could be used by other research laboratories in this field.