Site preference and local structural stability of Bi(III) substitution in hydroxyapatite using first-principles simulations.

Bismuth (Bi(III)) substitution in hydroxyapatite (HAp) lattice confers unique properties such as antibacterial, catalytic, radiosensitization, and conductive properties while preserving the innate bioactivity. Understanding the local structural changes upon Bi3+ substitution is essential for controlling the stability and optimizing the properties of HAp. Despite numerous experimental studies, the precise substitution behaviors, such as site preference and structural stability, remain incompletely understood. In this study, the substitution behavior of Bi(III) into the HAp lattice with formula of Ca9Bi(PO4)6(O)(OH) was investigated via first-principles simulation by implementing density functional theory. Energy calculations showed that Bi3+ preferentially occupies the Ca(2) site with an energy difference of ∼0.02 eV per atom. Local structure analysis revealed higher bond population values and an oxygen coordination shift from 7 to 6 for the Ca(2) site, attributed to the greater covalent interactions and its flexible environment accommodating the bulky Bi3+ ion and its stereochemically active lone pair. This work provides the first comprehensive investigation on Bi3+ ion substitution site preference in HAp using first-principles simulations.

Interfacial Modeling of Fibrinogen Adsorption onto LiNbO3 Single Crystal-Single Domain Surfaces.

For the development of next-generation protein-based biosensor surfaces, it is important to understand how functional proteins, such as fibrinogen (FBG), interact with polar substrate surfaces in order to prepare highly sensitive points of medical care diagnostics. FBG, which is a fibrous protein with an extracellular matrix, has both positively and negatively charged regions on its 3-dimensional surface, which makes interpreting how it effectively binds to polarized surfaces challenging. In this study, single-crystal LiNbO3 (LNO) substrates that have surface charges were used to investigate the adsorption of FBG protruding polar fragments on the positively and negatively charged LNO surfaces. We performed a combination of experiments and multi-scale molecular modeling to understand the binding of FBG in vacuum and water-solvated surfaces of LNO. XPS measurements showed that the FBG adsorption on LNO increased with increment in solution concentration on surfaces independent of charges. Multi-scale molecular modeling employing Quantum Mechanics, Monte Carlo, and Molecular Mechanics addressed the phenomenon of FBG fragment bonding on LNO surfaces. The binding simulation validated the experimental observation using zeta potential measurements which showed presence of solvated medium influenced the adsorption phenomenon due to the negative surface potential.

Synthesis of luminescent nanoporous silica spheres functionalized with folic acid for targeting to cancer cells.

Luminescent europium(III)-doped nanoporous silica nanospheres (Eu:NPS) were successfully synthesized, and a folate N-hydroxysuccinimidyl ester (FA-NHS) molecule as a targeting ligand for cancer cells was immobilized on the nanosphere surfaces through mediation of the 3-aminopropyltriethoxysilane (APTES) adlayer. The ordered nanopores were preserved by the immobilization, and the specific surface area decreased only with the APTES immobilization, suggesting that the FA-NHS was predominantly immobilized on the outer surface of the nanopores. The photoluminescence of the nanospheres functionalized with folic acid (FA) exhibited a characteristic peak due to the interactions (e.g., energy transfer) between FA and Eu(3+), and further the orange luminescence could be clearly detected by fluorescence microscopy in air and water. Furthermore, the nanospheres highly dispersed in cell culture medium exhibited nontoxicity in the cellular proliferation stages of the Hela cancer cells and NIH3T3 fibroblasts and specifically bind to the Hela cells. The nanospheres after the binding and uptake also showed intense luminescence from the outer/inner cell surfaces for the culture time of 4 days. Therefore, the luminescent FA-functionalized Eu:NPS nanospheres could be used for specific targeting and imaging abilities for cancer cells.

Photoluminescence and doping mechanism of theranostic Eu3+/Fe3+ dual-doped hydroxyapatite nanoparticles.

Theranostic nanoparticles currently have been regarded as an emerging concept of 'personalized medicine' with diagnostic and therapeutic dual-functions. Eu3+ doped hydroxyapatite (HAp) has been regarded as a promising fluorescent probe for in vivo imaging applications. Additionally, substitution of Ca2+ with Fe3+ in HAp crystal may endow the capability of producing heat upon exposure to a magnetic field. Here we report a preliminary study of doping mechanism and photoluminescence of Eu3+ and Fe3+ doped HAp nanoparticles (Eu/Fe:HAp). HAp with varied concentration of Eu3+ and Fe3+ doping are presented as Eu(10 mol%):HAp, Eu(7 mol%)-Fe(3 mol%):HAp, Eu(5 mol%)-Fe(5 mol%):HAp, Eu(3 mol%)-Fe(7 mol%):HAp, and Fe(10 mol%):HAp in the study. The results showed that the HAp particles, in nano-size with rod-like morphology, were successfully doped with Eu3+ and Fe3+, and the particles can be well suspended in cell culture medium. Photoluminescence analysis revealed that particles have prominent emissions at 536 nm, 590 nm, 615 nm, 650 nm and 695 nm upon excitation at a wavelength of 397 nm. Moreover, these Eu/Fe:HAp nanoparticles belonged to B-type carbonated HAp, which has been considered an effective biodegradable and biocompatible drug/gene carrier in biological applications.

Folic acid-mediated enhancement of the diagnostic potential of luminescent europium-doped hydroxyapatite nanocrystals for cancer biomaging.

Early and accurate cancer diagnosis is crucial for improving patient survival rates. Luminescent nanoparticles have emerged as a promising tool in fluorescence bioimaging for cancer diagnosis. To enhance diagnostic accuracy, ligands promoting endocytosis into cancer cells are commonly incorporated onto nanoparticle surfaces. Folic acid (FA) is one such ligand, known to specifically bind to folate receptors (FR) overexpressed in various cancer cells such as cervical and ovarian carcinoma. Therefore, surface modification of luminescent nanoparticles with FA can enhance both luminescence efficiency and diagnostic accuracy. In this study, luminescent europium-doped hydroxyapatite (EuHAp) nanocrystals were prepared via hydrothermal method and subsequently modified with (3-Aminopropyl)triethoxysilane (APTES) followed by FA to target FR-positive human cervical adenocarcinoma cell line (HeLa) cells. The sequential grafting of APTES and then FA formed a robust covalent linkage between the nanocrystals and FA. Rod-shaped FA-modified EuHAp nanocrystals, approximately 100 nm in size, exhibited emission peaks at 589, 615, and 650 nm upon excitation at 397 nm. Despite a reduction in photoluminescence intensity following FA modification, fluorescence microscopy revealed a remarkable 120-fold increase in intensity compared to unmodified EuHAp, attributed to the enhanced uptake of FA-modified EuHAp. Additionally, confocal microscope observations confirmed the specificity and the internalization of FA-modified EuHAp nanocrystals in HeLa cells. In conclusion, the modification of EuHAp nanocrystals with FA presents a promising strategy to enhance the diagnostic potential of cancer bioimaging probes.

Adsorption of l-buthionine sulfoximine on Bi(III) and Eu(III) co-substituted hydroxyapatite nanocrystals for enhancing radiosensitization effects.

Cancer theranostics combines therapeutic and diagnostic capabilities into a single system to treat cancer efficiently. Biocompatible nanomaterials can be engineered to exhibit cancer theranostic functions, for instance radiosensitization and photoluminescence. In this study, trivalent Bi and Eu ions were co-substituted into the lattice of hydroxyapatite (Bi(III):Eu(III) HAp) to develop a cancer theranostic nanocrystal. Bi provides radiosensitization capabilities while Eu imparts photoluminescence properties. To complement the radiotherapeutic function, l-buthionine sulfoximine (l-BSO) was adsorbed onto the nanocrystal surface. l-BSO inhibits the biosynthesis of cellular antioxidants, which can enhance radiosensitization effects. The Bi(III):Eu(III) HAp nanocrystals were prepared via a hydrothermal method. Structural and compositional analyses showed that both Bi and Eu ions were substituted into the HAp lattice. l-BSO was adsorbed onto the surface via electrostatic interactions between the charged carboxyl and amino groups of l-BSO and the surface ions of the nanocrystals. The adsorption followed the Langmuir isotherm model, implying a homogeneous monolayer adsorption. The l-BSO adsorbed Bi(III):Eu(III) HAp nanocrystals were found to have negligible cytotoxicity except the setting with l-BSO adsorbed amounts of 0.44 µmol/m2. This l-BSO amount was found to be high enough to elicit cytotoxicity due to l-BSO being released and causing excessive antioxidant depletion. Gamma ray irradiation clearly activated the cytotoxicity of the samples and increased the cell death rate, confirming radiosensitization abilities. At a constant amount of nanocrystals, the cell death rate increases with l-BSO concentration. This indicates that l-BSO can enhance the radiosensitization effect of the Bi(III):Eu(III) HAp nanocrystals.

Hierarchical and urchin-like chitosan/hydroxyapatite microspheres as drug-laden cell carriers.

Biopolymer/hydroxyapatite (HAp) composites are one type of the most promising materials for a variety of biomedical applications. In this study, hierarchical and urchin-like chitosan/HAp nanowire (HU-CS/HAp NW) microspheres were for the first time synthesized by in situ hydrothermal treatment of chitosan/HAp (CS/HAp) microspheres in the acetic acid solution. The results indicate that HU-CS/HAp NW microspheres were spherical in morphology with a diameter of 100-300 µm. Their surface was mainly constructed by numerous HAp NWs with the diameter of 80-120 nm and showed a hierarchical and urchin-like nanofibrous architecture. It was found that the acidic hydrothermal treatment caused an in situ conversion of HAp NPs to HAp NWs. In vitro biocompatible evaluation indicates that HU-CS/HAp NW microspheres showed an enhanced cell attachment and proliferation due to the presence of hierarchical and urchin-like architecture. Furthermore, HU-CS/HAp NW microspheres showed a good adsorption capacity for tetracycline hydrochloride (model drug, one of the most representative antibiotics) with a higher adsorption capacity than CS/HAp microspheres and well maintained their antibacterial efficacy to inhibit the growth of bacteria: Escherichia coli and Staphylococcus aureus. Thus, the present HU-CS/HAp NW microspheres would be applicable as novel drug-laden cell carriers.

Cyclic stretch modulates the cell morphology transition under geometrical confinement by covalently immobilized gelatin.

Fibroblasts geometrically confined by photo-immobilized gelatin micropatterns were subjected to cyclic stretch on the silicone elastomer. By using covalently micropatterned surfaces, the cell morphologies such as cell area and length were quantitatively investigated under a cyclic stretch for 20 hours. The mechanical forces did not affect the cell growth but significantly altered the cellular morphology on both non-patterned and micropatterned surfaces. It was found that cells on non-patterns showed increasing cell length and decreasing cell area under the stretch. The width of the strip micropatterns provided a different extent of contact guidance for fibroblasts. The highly extended cells on the 10 µm pattern under static conditions would perform a contraction behavior once treated by cyclic stretch. In contrast, cells with a low extension on the 2 µm pattern kept elongating according to the micropattern under the cyclic stretch. The vertical stretch induced an increase in cell area and length more than the parallel stretch in both the 10 µm and 2 µm patterns. These results provided new insights into cell behaviors under geometrical confinement in a dynamic biomechanical environment and may guide biomaterial design for tissue engineering in the future.

Visualized procollagen Iα1 demonstrates the intracellular processing of propeptides.

The processing of type I procollagen is essential for fibril formation; however, the steps involved remain controversial. We constructed a live cell imaging system by inserting fluorescent proteins into type I pre-procollagen α1. Based on live imaging and immunostaining, the C-propeptide is intracellularly cleaved at the perinuclear region, including the endoplasmic reticulum, and subsequently accumulates at the upside of the cell. The N-propeptide is also intracellularly cleaved, but is transported with the repeating structure domain of collagen into the extracellular region. This system makes it possible to detect relative increases and decreases in collagen secretion in a high-throughput manner by assaying fluorescence in the culture medium, and revealed that the rate-limiting step for collagen secretion occurs after the synthesis of procollagen. In the present study, we identified a defect in procollagen processing in activated hepatic stellate cells, which secrete aberrant collagen fibrils. The results obtained demonstrated the intracellular processing of type I procollagen, and revealed a link between dysfunctional processing and diseases such as hepatic fibrosis.

Transfection efficiency for size-separated cells synchronized in cell cycle by microfluidic device.

Non-viral system generally demonstrates less efficacious in transgene delivery than viral system; however it represents a safer alternative to viral system. In this study, transfection efficiency for human hepatocellular liver carcinoma cells synchronized in cell cycle at G0/G1 phase, which was sorted in size with a microfluidic device based on hydrodynamic filtration, was investigated by using a reverse transfection method. The synchronized cells were recovered at the yield of 80% from the micro-channel, and green fluorescent protein gene encoding plasmid mixed with lipofectoamine was transfected. The transfection efficiency of the cells at G0/G1 phase was 1.8 times higher than non-synchronized cells. The manipulation of cell cycle status could increase transfection efficiency in non-viral system, indicating size-based cell cycle synchronization is a powerful tool as a noninvasive method for bioscience and biotechnology.

Effect of interfacial proteins on osteoblast-like cell adhesion to hydroxyapatite nanocrystals.

A quartz crystal microbalance with dissipation (QCM-D) technique was employed to detecting the protein adsorption and subsequent osteoblast-like cell adhesion to hydroxyapatite (HAp) nanocrystals. The interfacial phenomena with the preadsorption of three proteins (albumin (BSA), fibronectin (Fn), and collagen (Col)), the subsequent adsorption of fetal bovine serum (FBS), and the adhesion of the cells were investigated. The QCM-D measured the frequency shift (Δf) and dissipation energy shift (ΔD), and the viscoelastic properties of the adlayers were evaluated using ΔD-Δf plot and Voigt-based viscoelastic model. The Col adsorption significantly showed higher Δf, ΔD, elasticity, and viscosity values as compared to the BSA and Fn adsorption, and the subsequent FBS adsorption depended on the preadsorbed proteins. The ΔD-Δf plot of the cell adhesion also showed a different behavior depending on the surfaces, and the Fn- and Col-modified surfaces showed the rapid mass and ΔD changes by forming the viscous interfacial layers with cell adhesion, indicating that the processes were affected by the cellular reaction through the extracellular matrix (ECM) proteins. The confocal laser scanning microscope images of adherent cells showed a different morphology and pseudopod on the surfaces. The cells adhered to the surfaces modified with the Fn and Col had significantly uniaxially expanded shapes and fibrous pseudopods, and those modified with the BSA had a round shape. Therefore, the different cell-protein interactions would cause the arrangement of the ECM and the cytoskeleton changes at the interfaces, and these phenomena were successfully detected by the QCM-D and Voigt-based model.

Detection of interfacial phenomena with osteoblast-like cell adhesion on hydroxyapatite and oxidized polystyrene by the quartz crystal microbalance with dissipation.

The adhesion process of osteoblast-like cells on hydroxyapatite (HAp) and oxidized polystyrene (PSox) was investigated using a quartz crystal microbalance with dissipation (QCM-D), confocal laser scanning microscope (CLSM), and atomic force microscope (AFM) techniques in order to clarify the interfacial phenomena between the surfaces and cells. The interfacial viscoelastic properties (shear viscosity (η(ad)), elastic shear modulus (µ(ad)), and tan δ) of the preadsorbed protein layer and the interface layer between the surfaces and cells were estimated using a Voigt-based viscoelastic model from the measured frequency (Δf) and dissipation shift (ΔD) curves. In the ΔD-Δf plots, the cell adhesion process on HAp was classified as (1) a mass increase only, (2) increases in both mass and ΔD, and (3) slight decreases in mass and ΔD. On PSox, only ΔD increases were observed, indicating that the adhesion behavior depended on the surface properties. The interfacial µ(ad) value between the material surfaces and cells increased with the number of adherent cells, whereas η(ad) and tanδ decreased slightly, irrespective of the surface. Thus, the interfacial layer changed the elasticity to viscosity with an increase in the number. The tan δ values on HAp were higher than those on PSox and exceeded 1.0. Furthermore, the pseudopod-like structures of the cells on HAp had periodic stripe patterns stained with a type I collagen antibody, whereas those on PSox had cell-membrane-like structures unstained with type I collagen. These results indicate that the interfacial layers on PSox and HAp exhibit elasticity and viscosity, respectively, indicating that the rearrangements of the extracellular matrix and cytoskeleton changes cause different cell-surface interactions. Therefore, the different cell adhesion process, interfacial viscoelasticity, and morphology depending on the surfaces were successfully monitored in situ and evaluated by the QCM-D technique combined with other techniques.

Minerals and aligned collagen fibrils in tilapia fish scales: structural analysis using dark-field and energy-filtered transmission electron microscopy and electron tomography.

The mineralized structure of aligned collagen fibrils in a tilapia fish scale was investigated using transmission electron microscopy (TEM) techniques after a thin sample was prepared using aqueous techniques. Electron diffraction and electron energy loss spectroscopy data indicated that a mineralized internal layer consisting of aligned collagen fibrils contains hydroxyapatite crystals. Bright-field imaging, dark-field imaging, and energy-filtered TEM showed that the hydroxyapatite was mainly distributed in the hole zones of the aligned collagen fibrils structure, while needle-like materials composed of calcium compounds including hydroxyapatite existed in the mineralized internal layer. Dark-field imaging and three-dimensional observation using electron tomography revealed that hydroxyapatite and needle-like materials were mainly found in the matrix between the collagen fibrils. It was observed that hydroxyapatite and needle-like materials were preferentially distributed on the surface of the hole zones in the aligned collagen fibrils structure and in the matrix between the collagen fibrils in the mineralized internal layer of the scale.

Competitive adsorption of fibronectin and albumin on hydroxyapatite nanocrystals.

Competitive adsorption of two-component solutions containing fibronectin (Fn) and albumin (Ab) on hydroxyapatite (HAp) nanocrystals was analyzed in situ using the quartz crystal microbalance with dissipation (QCM-D) technique. Adsorption of the one-component protein (Fn or Ab) and the two-component proteins adjusted to different molar ratios of Fn to Ab at a fixed Fn concentration was investigated. The frequency shift (Δf; Hz) and the dissipation energy shift (ΔD) were measured with the QCM-D technique, and the viscoelastic changes of adlayers were evaluated by the saturated ΔD/Δf value and the Voigt-based viscoelastic model. For the adsorption of the one-component protein, the Fn adlayer showed a larger mass and higher viscoelasticity than the Ab adlayer, indicating the higher affinity of Fn on HAp. For the adsorption of the two-component proteins, the viscoelastic properties of the adlayers became elastic with increase in Ab concentration, whereas the adsorption mass was similar to that of Fn in the one-component solution regardless of the Ab concentration. The specific binding mass of the Ab antibody to the adlayers increased with increase in Ab concentration, whereas that of the Fn antibody decreased. Therefore, Fn preferentially adsorbs on HAp and Ab subsequently interacts with the adlayers, indicating that the interfacial viscoelasticity of the adlayers was dominated by the interaction between Fn and Ab.

BMP-2-loaded silica nanotube fibrous meshes for bone generation.

Silica nanotube fibrous meshes were fabricated as multiple functional matrices for both delivering bone morphological protein-2 (BMP-2) and supporting osteoblast attachment and proliferation. The meshes were fabricated via a collagen-templated sol-gel route and consisted of tubular silica with open ends. BMP-2 was loaded to the meshes by soaking in BMP-2 solution. The meshes effectively enabled the attachment and proliferation of osteoblast MC3T3-E1 cells and delivered bioactive BMP-2 to stimulate cell differentiation. These results demonstrate the potential use of the meshes in bone generation applications.

Local administration and enhanced release of bone metabolic antibodies from hydroxyapatite/chondroitin sulfate nanocomposite microparticles using zinc cations.

As a local delivery carrier of bone metabolic proteins, we have previously reported hydroxyapatite/chondroitin sulfate composite microparticles (HAp/ChS) and their formulation method using zinc cations (Zn), and the in vitro release properties of proteins from the microparticles. Herein, we report the release properties of model antibodies such as immunoglobulin (IgG), human IgG (hIgG), and denosumab (Dmab) from HAp/ChS using this formulation method. Adding Zn in the formulation of IgG loaded with HAp/ChS microparticles enhanced the release of antibodies from HAp/ChS in phosphate buffer saline. In addition, the biological activity of Dmab released from HAp/ChS formulated with Zn was significantly higher than that without Zn. These results suggest a possible beneficial effect on the treatment for local bone diseases. The sclerostin monoclonal antibody (Sclmab) promotes fracture healing. We prepared HAp/ChS microparticles loaded with Sclmab and locally administered the microparticles into a drilled hole in the distal femoral bone of young rats. After three weeks, the area of the newly formed osteoid around the drilled hole where HAp/ChS loaded with Sclmab and Zn was locally administered was significantly higher than that observed in the control group (normal saline). Thus, HAp/ChS microparticles and the formulation method of monoclonal antibodies using Zn could be useful in the treatment of local bone diseases.

Magnetic Field Alignment, a Perspective in the Engineering of Collagen-Silica Composite Biomaterials.

Major progress in the field of regenerative medicine is expected from the design of artificial scaffolds that mimic both the structural and functional properties of the ECM. The bionanocomposites approach is particularly well fitted to meet this challenge as it can combine ECM-based matrices and colloidal carriers of biological cues that regulate cell behavior. Here we have prepared bionanocomposites under high magnetic field from tilapia fish scale collagen and multifunctional silica nanoparticles (SiNPs). We show that scaffolding cues (collagen), multiple display of signaling peptides (SiNPs) and control over the global structuration (magnetic field) can be combined into a unique bionanocomposite for the engineering of biomaterials with improved cell performances.

Erratum: Debons et al. Magnetic Field Alignment, a Perspective in the Engineering of Collagen-Silica Composite Biomaterials. Biomolecules 2021, 11, 749.

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Recent advances in bioprinting technologies for engineering different cartilage-based tissues.

Inadequate self-repair and regenerative efficiency of the cartilage tissues has motivated the researchers to devise advanced and effective strategies to resolve this issue. Introduction of bioprinting to tissue engineering has paved the way for fabricating complex biomimetic engineered constructs. In this context, the current review gears off with the discussion of standard and advanced 3D/4D printing technologies and their implications for the repair of different cartilage tissues, namely, articular, meniscal, nasoseptal, auricular, costal, and tracheal cartilage. The review is then directed towards highlighting the current stem cell opportunities. On a concluding note, associated critical issues and prospects for future developments, particularly in this sphere of personalized medicines have been discussed.

Rattle-type Fe(3)O(4)@SiO(2) hollow mesoporous spheres as carriers for drug delivery.

Rattle-type Fe(3)O(4)@SiO(2) hollow mesoporous spheres with different particle sizes, different mesoporous shell thicknesses, and different levels of Fe(3)O(4) content are prepared by using carbon spheres as templates. The effects of particle size and concentration of Fe(3)O(4)@SiO(2) hollow mesoporous spheres on cell uptake and their in vitro cytotoxicity to HeLa cells are evaluated. The spheres exhibit relatively fast cell uptake. Concentrations of up to 150 microg mL(-1) show no cytotoxicity, whereas a concentration of 200 microg mL(-1) shows a small amount of cytotoxicity after 48 h of incubation. Doxorubicin hydrochloride (DOX), an anticancer drug, is loaded into the Fe(3)O(4)@SiO(2) hollow mesoporous spheres, and the DOX-loaded spheres exhibit a somewhat higher cytotoxicity than free DOX. These results indicate the potential of Fe(3)O(4)@SiO(2) hollow mesoporous spheres for drug loading and delivery into cancer cells to induce cell death.