Enzyme-free and rapid colorimetric analysis of osteopontin via triple-helix aptamer probe coupled with catalytic hairpin assembly reaction.

BACKGROUND: Osteopontin (OPN) is closely associated with tumorigenesis, growth, invasion, and immune escape and it serves as a plasma biomarker for hepatocellular carcinoma (HCC). Nevertheless, the accurate and rapid detection of low-abundance OPN still poses significant challenges. Currently, the majority of protein detection methods rely heavily on large precision instruments or involve complex procedures. Therefore, developing a simple, enzyme-free, rapid colorimetric analysis method with high sensitivity is imperative. RESULTS: In this study, we have developed a portable colorimetric biosensor by integrating the triple-helix aptamer probe (THAP) and catalytic hairpin assembly (CHA) strategy, named as T-CHA. After binding to the OPN, the trigger probe can be released from THAP, then initiates the CHA reaction and outputs the signal through the formation of a G-quadruplex/Hemin DNAzyme with horseradish peroxidase-like activity. Consequently, this colorimetric sensor achieves visual free-labeled detection without additional fluorophore modification and allows for accurate quantification by measuring the optical density of the solution at 650 nm. Under optimal conditions, the logarithmic values of various OPN concentrations exhibit satisfactory linearity in the range of 5 pg mL-1 to 5 ng mL-1, with a detection limit of 2.04 pg mL-1. Compared with the widely used ELISA strategy, the proposed T-CHA strategy is rapid (∼105 min), highly sensitive, and cost-effective. SIGNIFICANCE: The T-CHA strategy, leveraging the low background leakage of THAP and the high catalytic efficiency of CHA, has been successfully applied to the detection of OPN in plasma, demonstrating significant promise for the early diagnosis of HCC in point-of-care testing. Given the programmability of DNA and the universality of T-CHA, it can be readily modified for analyzing other useful tumor biomarkers.

The impact of hydroxyapatite crystal structures and protein interactions on bone's mechanical properties.

Hydroxyapatite (HAP) constitutes the primary mineral component of bones, and its crystal structure, along with the surface interaction with proteins, significantly influences the outstanding mechanical properties of bone. This study focuses on natural hydroxyapatite, constructing a surface model with calcium vacancy defects. Employing a representative model of aspartic acid residues, we delve into the adsorption mechanism on the crystal surface and scrutinize the adsorption forms of amino acid residues on HAP and calcium-deficient hydroxyapatite (CDHA) surfaces. The research also explores the impact of different environments on adsorption energy. Furthermore, a simplified sandwich structure of crystal-polypeptide-crystal is presented, analyzing the distribution of amino acid residue adsorption sites on the crystal surface of the polypeptide fragment. This investigation aims to elucidate how the stick-slip mechanism of polypeptide molecules on the crystal surface influences the mechanical properties of the system. By uncovering the interface mechanical behavior between HAP and osteopontin peptides, this article offers valuable theoretical insights for the construction and biomimetic design of biocomposites.

Thrombin cleavage of osteopontin initiates osteopontin's tumor-promoting activity.

BACKGROUND: Osteopontin (OPN) is a multifunctional proinflammatory matricellular protein overexpressed in multiple human cancers and associated with tumor progression and metastases. Thrombin cleavage of OPN reveals a cryptic binding site for α4 ß1 and α9 ß1 integrins. METHODS: Thrombin cleavage-resistant OPNR153A knock-in (OPN-KI) mice were generated and compared to OPN deficient mice (OPN-KO) and wild type (WT) mice in their ability to support growth of melanoma cells. Flow cytometry was used to analyze tumor infiltrating leukocytes. RESULTS: OPN-KI mice engineered with a thrombin cleavage-resistant OPN had reduced B16 melanoma growth and fewer pulmonary metastases than WT mice. The tumor suppression phenotype of the OPN-KI mouse was identical to that observed in OPN-KO mice and was replicated in WT mice by pharmacologic inhibition of thrombin with dabigatran. Tumors isolated from OPN-KI mice had increased tumor-associated macrophages with an altered activation phenotype. Immunodeficient OPN-KI mice (NOG-OPN-KI) or macrophage-depleted OPN-KI mice did not exhibit the tumor suppression phenotype. As B16 cells do not express OPN, thrombin-cleaved fragments of host OPN suppress host antitumor immune response by functionally modulating the tumor-associated macrophages. YUMM3.1 cells, which express OPN, showed less tumor suppression in the OPN-KI and OPN-KO mice than B16 cells, but its growth was suppressed by dabigatran similar to B16 cells. CONCLUSIONS: Thrombin cleavage of OPN, derived from the host and the tumor, initiates OPN's tumor-promoting activity in vivo.

Osteopontin on the Dental Implant Surface Promotes Direct Osteogenesis in Osseointegration.

After dental implantation, osteopontin (OPN) is deposited on the hydroxyapatite (HA) blasted implant surface followed by direct osteogenesis, which is significantly disturbed in Opn-knockout (KO) mice. However, whether applying OPN on the implant surface promotes direct osteogenesis remains unclarified. This study analyzed the effects of various OPN modified protein/peptides coatings on the healing patterns of the bone-implant interface after immediately placed implantation in the maxilla of four-week-old Opn-KO and wild-type (WT) mice (n = 96). The decalcified samples were processed for immunohistochemistry for OPN and Ki67 and tartrate-resistant acid phosphatase histochemistry. In the WT mice, the proliferative activity in the HA binding peptide-OPN mimic peptide fusion coated group was significantly higher than that in the control group from day 3 to week 1, and the rates of OPN deposition and direct osteogenesis around the implant surface significantly increased in the recombinant-mouse-OPN (rOPN) group compared to the Gly-Arg-Gly-Asp-Ser peptide group in week 2. The rOPN group achieved the same rates of direct osteogenesis and osseointegration as those in the control group in a half period (week 2). None of the implant surfaces could rescue the direct osteogenesis in the healing process in the Opn-KO mice. These results suggest that the rOPN coated implant enhances direct osteogenesis during osseointegration following implantation.

Energy dissipation of osteopontin at a HAp mineral interface: Implications for bone biomechanics.

Osteopontin (OPN) is a one of the most abundant non-collagenous proteins in the bone's organic matrix. OPN is responsible for mediating bonding at mineral interfaces in the extrafibrillar space and recent evidence shows that it is a major contributor to bone's fracture resistance. While several experimental studies have identified an important role for calcium ions in mediating energy dissipation in OPN protein networks, the underlying molecular mechanisms remain largely unknown. In the current study, the role of calcium ions on energy dissipation at OPN interface with hydroxyapatite (HAp) as the main bone mineral was investigated. For the first time, the three-dimensional structure of OPN proteins were predicted, and it was found that calcium ions greatly influenced the final protein configuration and energy dissipation performance. Under small deformation, the compact cOPN structure, resulting from calcium ions presence, facilitated greater energy dissipation through sacrificial bond breaking and mechanisms mediated by the surface-bound calcium. At larger deformation, the compact structure also enabled cOPN to dissipate higher energy. Moreover, it was found that phosphorylation of OPN played an important role in energy dissipation. While previous studies have shown that OPN dissipated energy by forming aggregate networks, this study also showed that network formation is not necessary and that individual OPN proteins can dissipate large amounts of energy at HAp interfaces.

Distinctive Prognostic Value and Cellular Functions of Osteopontin Splice Variants in Human Gastric Cancer.

BACKGROUND: Osteopontin (OPN) splice variants are identified as predictors of tumour progression and therapeutic resistance in certain types of solid tumours. However, their roles in gastric cancer (GC) remain poorly characterized. The current study sought to assess the prognostic value of the three OPN splice variants (namely OPN-a, OPN-b, and OPN-c) in gastric cancer and their potential functions within gastric cancer cells. METHODS: RNA extraction and reverse transcription were performed using our clinical cohort of gastric carcinomas and matched normal tissues (n = 324 matched pairs). Transcript levels were determined using real-time quantitative PCR. Three OPN splice variants overexpressed cell lines were created from the gastric cancer cell line HGC-27. Subsequently, biological functions, including cell growth, adhesion, migration, and invasion, were studied. The potential effects of OPN isoforms on cisplatin and 5-Fu were evaluated by detecting cellular reactive oxygen species (ROS) levels in the HGC-27-derived cell lines. RESULTS: Compared with normal tissues, the expression levels of three splice variants were all elevated in gastric cancer tissues in an order of OPN-a > OPN-b > OPN-c. The OPN-a level significantly increased with increasing TNM staging and worse clinical outcome. There appeared to be a downregulation for OPN-c in increasing lymph node status (p < 0.05), increasing TNM staging, and poor differentiation. High levels of OPN-a and OPN-b were correlated with short overall survival and disease-free survival of gastric cancer patients. However, the low expression of OPN-c was significantly associated with a poor prognosis. Functional analyses further showed that ectopic expression of OPN-c suppressed in vitro proliferation, adhesiveness, migration, and invasion properties of HGC-27 cells, while the opposite role was seen for OPN-a. Cellular ROS detection indicated that OPN-a and OPN-c significantly promoted ROS production after treatment with 5-Fu comparing to OPN-vector, while only OPN-a markedly induced ROS production after treatment with cisplatin. CONCLUSION: Our results suggest that OPN splice variants have distinguished potential to predict the prognosis of gastric cancer. Three OPN variants exert distinctive functions in gastric cancer cells. Focusing on specific OPN isoforms could be a novel direction for developing diagnostic and therapeutic approaches in gastric cancer.

Intrinsically Disordered Osteopontin Fragment Orders During Interfacial Calcium Oxalate Mineralization.

Calcium oxalate (CaC2 O4 ) is the major component of kidney stone. The acidic osteopontin (OPN) protein in human urine can effectively inhibit the growth of CaC2 O4 crystals, thereby acting as a potent stone preventer. Previous studies in bulk solution all attest to the importance of binding and recognition of OPN at the CaC2 O4 mineral surface, yet molecular level insights into the active interface during CaC2 O4 mineralization are still lacking. Here, we probe the structure of the central OPN fragment and its interaction with Ca2+ and CaC2 O4 at the water-air interface using surface-specific non-linear vibrational spectroscopy. While OPN peptides remain largely disordered in solution, our results reveal that the bidentate binding of Ca2+ ions refold the interfacial peptides into well-ordered and assembled ß-turn motifs. One critical intermediate directs mineralization by releasing structural freedom of backbone and binding side chains. These insights into the mineral interface are crucial for understanding the pathological development of kidney stones and possibly relevant for calcium oxalate biomineralization in general.

Structural Constraint of Osteopontin Facilitates Efficient Binding to CD44.

Since the original description in 1996, the interaction between the cytokine osteopontin (OPN) and the homing receptor CD44 has been extensively studied in cancer, inflammation, bone remodeling, and various other conditions. Alternative splicing and extensive posttranslational modifications by both binding partners, as well as the possibility for lateral recruitment of additional membrane receptors or soluble co-ligands into a complex have left the exact molecular requirements for high-affinity OPN-CD44 binding unresolved. We now report that there is a moderate engagement between the unmodified molecules, which results in curved double-reciprocal plots for OPN titration, suggesting the existence of two binding sites or two binding conformations. Structural constraint of OPN, by immobilization or by addition of heparin, is required for its strong ligation of CD44. Prior literature provides evidence that heparin binding to OPN prompts the unfolding of a core element in the protein. This conformational adjustment may be essential for efficient CD44 interaction. The integrin α9ß1 seems to compete with the OPN-CD44 engagement, while the integrin αVß3 reflects additive binding, suggesting that the CD44 contact sites on OPN are downstream of the RGD motif but overlap with the SVVYGLR domain. Hyaluronate has no effect, placing the relevant domain on CD44 downstream of the N-terminus.

Binding Mode Characterization of Osteopontin on Hydroxyapatite by Solution NMR Spectroscopy.

Extracellular matrix glycoproteins play a major role in bone mineralization and modulation of osteogenesis. Among these, the intrinsically disordered protein osteopontin (OPN) is associated with the inhibition of formation, growth and proliferation of the bone mineral hydroxyapatite (HAP). Furthermore, post-translational modifications like phosphorylation can alter conformations and interaction properties of intrinsically disordered proteins (IDPs). Therefore, the actual interaction of OPN with a HAP surface on an atomic level and how this interaction is affected by phosphorylation is of great interest. Here, we study the interaction of full-length OPN on the surface of suspended HAP nanoparticles by solution NMR spectroscopy. We report the binding modes of this IDP and provide evidence for the influence of hyperphosphorylation on the binding character and an explanation for the differing roles in biomineralization. Our study moreover presents an easy and suitable option to measure interaction of nanoparticles in a stable suspension with full-length proteins.

Hyperphosphorylation of Human Osteopontin and Its Impact on Structural Dynamics and Molecular Recognition.

Protein phosphorylation is an abundant post-translational modification (PTM) and an essential modulator of protein functionality in living cells. Intrinsically disordered proteins (IDPs) are particular targets of PTM protein kinases due to their involvement in fundamental protein interaction networks. Despite their dynamic nature, IDPs are far from having random-coil conformations but exhibit significant structural heterogeneity. Changes in the molecular environment, most prominently in the form of PTM via phosphorylation, can modulate these structural features. Therefore, how phosphorylation events can alter conformational ensembles of IDPs and their interactions with binding partners is of great interest. Here we study the effects of hyperphosphorylation on the IDP osteopontin (OPN), an extracellular target of the Fam20C kinase. We report a full characterization of the phosphorylation sites of OPN using a combined nuclear magnetic resonance/mass spectrometry approach and provide evidence for an increase in the local flexibility of highly phosphorylated regions and the ensuing overall structural elongation. Our study emphasizes the simultaneous importance of electrostatic and hydrophobic interactions in the formation of compact substates in IDPs and their relevance for molecular recognition events.

Osteopontin Takes Center Stage in Chronic Liver Disease.

Osteopontin (OPN) was first identified in 1986. The prefix osteo- means bone; however, OPN is expressed in other tissues, including liver. The suffix -pontin means bridge and denotes the role of OPN as a link protein within the extracellular matrix. While OPN has well-established physiological roles, multiple "omics" analyses suggest that it is also involved in chronic liver disease. In this review, we provide a summary of the OPN gene and protein structure and regulation. We outline the current knowledge on how OPN is involved in hepatic steatosis in the context of alcoholic liver disease and non-alcoholic fatty liver disease. We describe the mechanisms whereby OPN participates in inflammation and liver fibrosis and discuss current research on its role in hepatocellular carcinoma and cholangiopathies. To conclude, we highlight important points to consider when doing research on OPN and provide direction for making progress on how OPN contributes to chronic liver disease.

Osteopontin regulates biomimetic calcium phosphate crystallization from disordered mineral layers covering apatite crystallites.

Details of apatite formation and development in bone below the nanometer scale remain enigmatic. Regulation of mineralization was shown to be governed by the activity of non-collagenous proteins with many bone diseases stemming from improper activity of these proteins. Apatite crystal growth inhibition or enhancement is thought to involve direct interaction of these proteins with exposed faces of apatite crystals. However, experimental evidence of the molecular binding events that occur and that allow these proteins to exert their functions are lacking. Moreover, recent high-resolution measurements of apatite crystallites in bone have shown that individual crystallites are covered by a persistent layer of amorphous calcium phosphate. It is therefore unclear whether non-collagenous proteins can interact with the faces of the mineral crystallites directly and what are the consequences of the presence of a disordered mineral layer to their functionality. In this work, the regulatory effect of recombinant osteopontin on biomimetic apatite is shown to produce platelet-shaped apatite crystallites with disordered layers coating them. The protein is also shown to regulate the content and properties of the disordered mineral phase (and sublayers within it). Through solid-state NMR atomic carbon-phosphorous distance measurements, the protein is shown to be located in the disordered phases, reaching out to interact with the surfaces of the crystals only through very few sidechains. These observations suggest that non-phosphorylated osteopontin acts as regulator of the coating mineral layers and exerts its effect on apatite crystal growth processes mostly from afar with a limited number of contact points with the crystal.

A Non-Invasive Nanoprobe for In Vivo Photoacoustic Imaging of Vulnerable Atherosclerotic Plaque.

Vulnerable atherosclerotic (AS) plaque is the major cause of cardiovascular death. However, clinical methods cannot directly identify the vulnerable AS plaque at molecule level. Herein, osteopontin antibody (OPN Ab) and NIR fluorescence molecules of ICG co-assembled Ti3 C2 nanosheets are reported as an advanced nanoprobe (OPN Ab/Ti3 C2 /ICG) with enhanced photoacoustic (PA) performance for direct and non-invasive in vivo visual imaging of vulnerable AS plaque. The designed OPN Ab/Ti3 C2 /ICG nanoprobes successfully realize obvious NIR fluorescence imaging toward foam cells as well as the vulnerable AS plaque slices. After intravenous injection of OPN Ab/Ti3 C2 /ICG nanoprobes into AS model mice, in vivo imaging results show a significantly enhanced PA signal in the aortic arch accumulated with vulnerable plaque, well indicating the remarkable feasibility of OPN Ab/Ti3 C2 /ICG nanoprobes to distinguish the vulnerable AS plaque. The proposed OPN Ab/Ti3 C2 /ICG nanoprobes not only overcome the clinical difficulty to differentiate vulnerable plaque, but also achieve the non-invasively specific in vivo imaging of vulnerable AS plaque at molecule level, greatly promoting the innovation of cardiovascular diagnosis technology.

Genetic and epigenetic regulation of osteopontin by cyclic adenosine 3' 5'-monophosphate in osteoblasts.

Osteopontin (OPN) is not only a marker of osteoblasts but it is also related to cancer progression and inflammation. The expression of OPN increases in response to inflammatory cytokines, hormones, and mechanical stress. Among them, cyclic-AMP (cAMP) elevating agents stimulate OPN expression in the presence of 1, 25-OH vitamin D3 (VD3). We aimed to clarify the mechanism by which cAMP enhances OPN expression in osteoblastic cells. The OPN promoter (-2335 to +76, OPNp2335) exerted a cell type specific response to forskolin (FK) and VD3. Sequential deletion analysis of OPNp revealed that the OPNp (-833 to +76) contained essential responsive regions to respond to cAMP signaling. In particular, both Vitamin D response element (VDRE, -758 to -743) and osteoblast-specific cis- acting element 2 (OSE2, -695 to -690) were essential for cAMP-mediated OPNp activity. The expression of vitamin D receptor (VDR), but not runt-related transcription factor 2 (Runx2), a nuclear receptor for OSE2, was induced by the treatment of the cells with FK. Although, VD3-induced OPNp activity was slightly enhanced in VDR-overexpressing osteoblasts, it reached the same level as that of osteoblasts induced by both VD3 and FK in the presence of histone deacetylase (HDAC) inhibitor. Moreover, we identified histone acetylation on the OPN promoter region by FK treatment. These results strongly suggest that OPNp activity is controlled by the cAMP signaling via genetic and epigenetic regulations.

Mesenchymal stem cell-derived microvesicles mediate BMP2 gene delivery and enhance bone regeneration.

A demineralized bone matrix (DBM) scaffold has good biocompatibility, low antigenicity, a natural porous structure and no cytotoxicity, and so it is an appropriate material for bone regeneration. However, osteoinductive growth factors are often removed during preparation, which destroys the osteoinductive capacity of the DBM scaffold. Biomaterials combined with gene therapy is a promising approach to effectively avoid this adverse side effect. This study develops a human bone morphogenetic protein 2 (hBMP2) gene-activated DBM scaffold to enhance the osteoinductive capacity of DBM and improve bone repair. Bone marrow mesenchymal stem cell (MSC)-derived microvesicles (MVs) were obtained, and polyethyleneimine (PEI) and human bone morphogenetic protein 2 (hBMP2) plasmids (phBMP2) were sequentially coated on the MVs by layer-by-layer (LBL) self-assembly to form an MVs-PEI/phBMP2 non-viral gene vector. Finally, the gene-activated scaffold (DBM/MVs-PEI/phBMP2) was prepared by loading MVs-PEI/phBMP2 onto a DBM scaffold. The experimental results show that the MVs-PEI/phBMP2 exhibits higher transfection efficiency and lower cytotoxicity to MSCs when the MVs/PEI weight ratio = 5, and could enhance the osteogenic differentiation of MSCs in vitro. Subcutaneous implantation into rats showed that the DBM/MVs-PEI/phBMP2 scaffold could efficiently enhance the deposition of: collagen fibers, osteocalcin, osteopontin and CD34 endogenous proteins. Rabbit femoral condyle defect experiments proved that the DBM/MVs-PEI/phBMP2 scaffold could significantly promote bone repair. This study presents a novel, highly efficient and low cytotoxicity gene delivery vector based on MVs. The gene-activated DBM scaffold based on MVs not only could promote bone formation but also angiogenesis, implying that this kind of gene-activated scaffold is a promising bone substitute material.

Osteopontin sequence modified mesoporous calcium silicate scaffolds to promote angiogenesis in bone tissue regeneration.

Sufficient blood supply remains the key issue to be addressed for an optimal performance of implanted bone tissue engineering scaffolds. Host vessel invasion is limited to a depth of only several hundred micrometers from the scaffold/host interface. In this study, an osteopontin sequenced polypeptide SVVYGLR was grafted into/onto mesoporous calcium silicate (MCS) and then 3D-printed into scaffolds. The peptide motifs can be accessed on the scaffold surfaces and released as well. In vitro studies of human umbilical vein endothelial cells (HUVECs) indicated enhanced cell adhesion and vascular-like structure formation on MCS-SVVYGLR scaffolds. At the same time, human bone marrow stromal cells (hBMSCs) showed enhanced osteogenic differentiation capability and higher expression levels of angiogenic genes and proteins as well. The results of in vivo radial defect repair tests of rabbits showed that more tubular vessels formed throughout the whole MCS-SVVYGLR scaffolds, and therefore, a more homogeneous new bone formation pattern was obtained on MCS-SVVYGLR scaffolds instead of a peripheral bone growth pattern on pure MCS scaffolds by Micro-CT and tissue staining techniques over 3 months. Relative gene and protein expressions in PI3K/AKT and ERK1/2 pathways suggested that the SVVYGLR motif on the MCS scaffold surface could initiate the PI3K/AKT signaling pathway and up-regulate ERK1/2 expression, which positively stimulated VEGF expression, to improve angiogenesis.

Evaluation of Bioactivities of the Bovine Milk Lactoferrin-Osteopontin Complex in Infant Formulas.

Human milk contains several bioactive proteins, including lactoferrin (LF) and osteopontin (OPN). These two proteins have been shown to form a complex, which shows increased bioactivities. Bovine LF and OPN can also form such a complex. We assessed bioactivities of the bovine LF-OPN complex (at molar ratios of LF:OPN = 3:1, 5:1, or 8:1) in a formula protein matrix, including LF, OPN, bovine whey protein hydrolysate, and α-lactalbumin. Our results show that the bovine LF-OPN complex together with formula proteins is resistant to in vitro digestion, stimulates intestinal cell proliferation (by 15-50%) and differentiation (by 30-50%), increases antibacterial activity (by 25-50%), and enhances intestinal immunity. The 3:1 ratio of LF to OPN exhibits the most potent effects, as compared with the other two ratios. In conclusion, adding bovine LF and OPN to infant formulas may result in increased stability of the two components and enhanced bioactivities, possibly improving outcomes in formula-fed infants.

Computational Nanomechanics of Noncollagenous Interfibrillar Interface in Bone.

The noncollagenous interfibrillar interface in bone provides the critical function of transferring loads among collagen fibrils and their bundles, with adhesive mechanisms at this site thus significantly contributing to the mechanical properties of bone. Motivated by the experimental observations and hypotheses, a computational study is presented to elucidate the critical roles of two major proteins at the nanoscale interfibrillar interface, that is, osteopontin (OPN) and osteocalcin (OC) in bone. This study reveals the extremely high interfacial toughness of the OPN/OC composite. The previously proposed hypothesis of sacrificial bonds in the extracellular organic matrix is tested, and the remarkable mechanical properties of the nanoscale bone interface are attributed to the collaborative interactions between the OPN and OC proteins.

Nanostructure of mouse otoconia.

Mammalian otoconia of the inner ear vestibular apparatus are calcium carbonate-containing mineralized structures critical for maintaining balance and detecting linear acceleration. The mineral phase of otoconia is calcite, which coherently diffracts X-rays much like a single-crystal. Otoconia contain osteopontin (OPN), a mineral-binding protein influencing mineralization processes in bones, teeth and avian eggshells, for example, and in pathologic mineral deposits. Here we describe mineral nanostructure and the distribution of OPN in mouse otoconia. Scanning electron microscopy and atomic force microscopy of intact and cleaved mouse otoconia revealed an internal nanostructure (~50 nm). Transmission electron microscopy and electron tomography of focused ion beam-prepared sections of otoconia confirmed this mineral nanostructure, and identified even smaller (~10 nm) nanograin dimensions. X-ray diffraction of mature otoconia (8-day-old mice) showed crystallite size in a similar range (73 nm and smaller). Raman and X-ray absorption spectroscopy - both methods being sensitive to the detection of crystalline and amorphous forms in the sample - showed no evidence of amorphous calcium carbonate in these mature otoconia. Scanning and transmission electron microscopy combined with colloidal-gold immunolabeling for OPN revealed that this protein was located at the surface of the otoconia, correlating with a site where surface nanostructure was observed. OPN addition to calcite growing in vitro produced similar surface nanostructure. These findings provide details on the composition and nanostructure of mammalian otoconia, and suggest that while OPN may influence surface rounding and surface nanostructure in otoconia, other incorporated proteins (also possibly including OPN) likely participate in creating internal nanostructure.

Osteopontin-loaded PLGA nanoparticles enhance the intestinal mucosal barrier and alleviate inflammation via the NF-κB signaling pathway.

Osteopontin is a multifunctional glycoprotein that is secreted by a variety of tissues or cells, but the role of osteopontin in the epithelial mucosal barrier has not been clearly established. We loaded osteopontin into hyaluronic acid-functionalized polymeric nanoparticles, which were administered by gavage to a colitis mouse model. The disease activity index, weight gain and colon length were calculated to assess the degree of symptoms. Epithelial permeability was measured using fluorescein isothiocyanate-conjugated dextran. The enzymatic activity of myeloperoxidase in the colon and inflammatory cytokines were assayed to assess the levels of inflammation. The histological appearance of the colon was observed by H&E staining. Tight junction proteins and signaling pathway proteins (NF-κB and phospho-NF-κB) were determined by western blotting. The resultant spherical osteopontin-loaded nanoparticles were characterized by the expected particle size (approximately 272.3 nm) and a slightly negative zeta potential (approximately -5.3 mV). Interestingly, we found that the osteopontin-loaded nanoparticles exerted remedial effects on colitis by both enhancing the intestinal barrier and alleviating inflammation in vivo according to the tested parameters. These results suggest that OPN plays a positive role in protecting the epithelial mucosal barrier and may be a therapeutic drug in gut homeostasis.