Synthesis, characterization, and evaluation of nano-hydroxyapatite based experimental calcium silicate cement as a root repair material / Síntesis, caracterización y evaluación de cemento experimental de silicato de calcio a base de nanohidroxiapatita como material de reparación radicular

Introduction: This study aimed to prepare a new root repair material including Portland cement, bismuth oxide, and nano-hydroxyapatite and analyze its physicochemical properties and its effects on the proliferation and differentiation of human dental pulp stem cells (hDPSCs). Material and Methods: Bismuth oxide as a radiopaque component and nano-hydroxyapatite particles were added to white Portland cement at 20% and 5% weight ratio, respectively. Characterization of the prepared cement was done using conventional methods. To examine the bioactivity of this new material, atomic absorption spectroscopy (AAS) was used for the investigation of the rate of calcium ions dissolution in simulated body fluid media. The viability of hDPSCs was assessed by an MTT assay after 1, 3 and 7 days. The odontogenic potential of this substance was evaluated by measuring alkaline phosphatase activity and alizarin red S staining. Results: Based on the bioactivity results, the cement presented high bio-activity, corroborating sufficiently with the calcium release patterns. The cell viability was significantly increased in new root repair material containing hydroxyapatite nanoparticles after 3 and 7 days (p<0.05). Conclusion: Moreover, alkaline phosphatase activity increased over 7 days in all experimental groups. The new cement containing nano-hydroxyapatite particles could be a good root repair material.

Objetivo: Este estudio tuvo como objetivo preparar un nuevo material de reparación de raíces que incluye cemento Portland, óxido de bismuto y nano-hidroxiapatita y analizar sus propiedades fisicoquímicas y sus efectos sobre la proliferación y diferenciación de células madre de pulpa dental humana. Material y Métodos: El óxido de bismuto como compo-nente radiopaco y las partículas de nano-hidroxiapatita se agregaron al cemento Portland blanco en una proporción en peso del 20 % y el 5 %, respectivamente. La caracterización del cemento preparado se realizó utilizando métodos con-vencionales. Para examinar la bioactividad de este nuevo material, se utilizó la espectroscopia de absorción atómica para investigar la velocidad de disolución de los iones de calcio en medio fluido corporal simulado. La viabilidad de las células madre de pulpa dental humana se evaluó mediante un ensayo MTT después de 1, 3 y 7 días. El potencial odontogénico de esta sustancia se evaluó midiendo la actividad de la fosfatasa alcalina y la tinción con rojo de alizarina S.Resultados: Con base en los resultados de bioactividad, el cemento presentó alta bioactividad, corroborando suficientemente con los patrones de liberación de calcio. La viabilidad celular aumentó significativamente en el nuevo material de reparación de raíces que contenía nanopartículas de hidroxiapatita después de 3 y 7 días (p<0,05). Conclusión: Además, la actividad de la fosfatasa alcalina aumentó durante 7 días en todos los grupos experimentales. El nuevo cemento que contiene partículas de nanohidroxiapatita podría ser un buen material de reparación radicular.

Construction of coral rod-like MoS2@HA nanowires hybrids for highly effective green antisepsis.

Antibacterial materials have been rapidly emerging as a primary component in the mitigation of bacterial pathogens, and green functional materials play a vital role in the antibacterial field. In this study, biocompatible hydroxyapatite nanowires (HANW) was used as a carrier, a coral rod-like nanowires hybrid of MoS2 and HANW (CR-MoS2@HANW) was synthesized via a facile two-step hydrothermal approach. After being characterized by scanning electron microscope (SEM), energy dispersive spectrometer (EDS), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), fourier transform infrared spectroscopy (FT-IR), Brunauer-Emmet-Teller (BET) and thermogravimetric (TG) analysis, the antibacterial activity and environmental compatibility were assessed. It was found that MoS2 nanosheets were in-situ assembled onto surface of HA nanowires, and the obtained nanohybrid exhibited excellent stability. CR-MoS2@HANW endowed a desirable long-term antibacterial activity against both gram-negative E. coli and gram-positive S. aureus. It was sufficient to inhibit the growth of bacteria within 72 h, and nanohybrids effectively promoted the growth of plants. In summary, the combination of MoS2 and HANW created a novel eco-friendly nanohybrids that could be applied as a promising multi-functional green antisepsis. And the CR-MoS2@HANW possessed enormous potential for biomedical applications.

Peptide-Decorated DNA Nanostructures Promote Site-Specific Hydroxyapatite Growth.

The guiding principle for mineralized tissue formation is that mineral growth occurs through the interaction of Ca2+ and phosphate ions with extracellular matrix (ECM) proteins. Recently, nanoengineered DNA structures have been proposed as mimics to ECM scaffolds. However, these principles have not been applied to mineralized tissues. Here, we describe DNA nanostructures, namely, a DNA nanotube and a DNA origami rectangle that are site specifically functionalized with a mineral-promoting "SSEE" peptide derived from ECM proteins present in mineralized tissues. In the presence of Ca2+ and phosphate ions (mineralizing conditions), site-specific calcium phosphate formation occurred on the DNA nanostructures. Amorphous calcium phosphate or hydroxyapatite was formed depending on the incubation time, shape of the DNA nanostructure, and amount of Ca2+ and phosphate ions present. The ability to design and control the growth of hydroxyapatite through nanoengineered scaffolds provides insights into the mechanisms that may occur during crystal nucleation and growth of mineralized tissues and can inspire mineralized tissue regeneration strategies.

Engineered Hydroxyapatite Nanoadjuvants with Controlled Shape and Aspect Ratios Reveal Their Immunomodulatory Potentials.

Hydroxyapatite (HAP) has been formulated as adjuvants in vaccines for human use. However, the optimal properties required for HAP nanoparticles to elicit adjuvanticity and the underlying immunopotentiation mechanisms have not been fully elucidated. Herein, a library of HAP nanorods and nanospheres was synthesized to explore the effect of the particle shape and aspect ratio on the immune responses in vitro and adjuvanticity in vivo. It was demonstrated that long aspect ratio HAP nanorods induced a higher degree of cell membrane depolarization and subsequent uptake, and the internalized particles elicited cathepsin B release and mitochondrial reactive oxygen species generation, which further led to pro-inflammatory responses. Furthermore, the physicochemical property-dependent immunostimulation capacities were correlated with their humoral responses in a murine hepatitis B surface antigen immunization model, with long aspect ratio HAP nanorods inducing higher antigen-specific antibody productions. Importantly, HAP nanorods significantly up-regulated the IFN-γ secretion and CD107α expression on CD8+ T cells in immunized mice. Further mechanistic studies demonstrated that HAP nanorods with defined properties exerted immunomodulatory effects by enhanced antigen persistence and immune cell recruitments. Our study provides a rational design strategy for engineered nanomaterial-based vaccine adjuvants.

Marine-Derived Biowaste Conversion into Bioceramic Membrane Materials: Contrasting of Hydroxyapatite Synthesis Methods.

Marine-derived biowaste increment is enormous, yet could be converted into valuable biomaterial, e.g., hydroxyapatite-based bioceramic. Bioceramic material possesses superiority in terms of thermal, chemical, and mechanical properties. Bioceramic material also has a high level of biocompatibility when projected into biological tissues. Tuning the porosity of bioceramic material could also provide benefits for bioseparation application, i.e., ultrafiltration ceramic membrane filtration for food and dairy separation processes. This work presents the investigation of hydroxyapatite conversion from crab-shells marine-based biowaste, by comparing three different methods, i.e., microwave, coprecipitation, and sol-gel. The dried crab-shells were milled and calcinated as calcium precursor, then synthesized into hydroxyapatite with the addition of phosphates precursors via microwave, coprecipitation, or sol-gel. The compound and elemental analysis, degree of crystallinity, and particle shape were compared. The chemical compounds and elements from three different methods were similar, yet the degree of crystallinity was different. Higher Ca/P ratio offer benefit in producing a bioceramic ultrafiltration membrane, due to low sintering temperature. The hydroxyapatite from coprecipitation and sol-gel methods showed a significant degree of crystallinity compared with that of the microwave route. However, due to the presence of Fe and Sr impurities, the secondary phase of Ca9FeH(PO4)7 was found in the sol-gel method. The secondary phase compound has high absorbance capacity, an advantage for bioceramic ultrafiltration membranes. Furthermore, the sol-gel method could produce a snake-like shape, compared to the oval shape of the coprecipitation route, another benefit to fabricate porous bioceramic for a membrane filter.

Hydroxyapatite grafted chitosan/laponite RD hydrogel: Evaluation of the encapsulation capacity, pH-responsivity, and controlled release behavior.

In this study, a pH-responsive drug carrier was developed for the controllable release of drugs in the gastric environment. Chitosan (CS), a pH-sensitive biopolymer, and laponite RD (LAP), a nano-clay with a high drug-loading capability, were used to design the new carrier. Hydroxyapatite (HA) was grafted into CS/LAP matrix through a simple co-precipitation technique to overcome the burst release of the CS/LAP. The structural analysis and swelling tests of products demonstrated that the co-precipitation method has led to the penetration of HA nanoparticles inside the CS/LAP matrix and occupying its hollow pores. Occupation of the empty pores can lead to the entrapment of drug molecules, thereby reducing the release rate. The nanocomposite showed a high loading capacity to ofloxacin as a drug model. The effects of HA content on release behavior of nanocomposite were investigated at simulated gastric (pH 1.2) and intestine (pH 7.4) environments. The results indicated a high pH sensitivity for CS/LAP/HA. HA grafting reduced the release rate remarkably regardless of pH. The release rate of CS/LAP/HA decreased by 44-63% in pH 1.2 and 41-51% in pH 7.4 compared to CS/LAP. Kinetic studies indicated that grafting the HA in CS/LAP has changed the drug release mechanism.

Mussel-Inspired Bisphosphonated Injectable Nanocomposite Hydrogels with Adhesive, Self-Healing, and Osteogenic Properties for Bone Regeneration.

Injectable hydrogels have received much attention because of the advantages of simulation of the natural extracellular matrix, microinvasive implantation, and filling and repairing of complex shape defects. Yet, for bone repair, the current injectable hydrogels have shown significant limitations such as the lack of tissue adhesion, deficiency of self-healing ability, and absence of osteogenic activity. Herein, a strategy to construct mussel-inspired bisphosphonated injectable nanocomposite hydrogels with adhesive, self-healing, and osteogenic properties is developed. The nano-hydroxyapatite/poly(l-glutamic acid)-dextran (nHA/PLGA-Dex) dually cross-linked (DC) injectable hydrogels are fabricated via Schiff base cross-linking and noncovalent nHA-BP chelation. The chelation between bisphosphonate ligands (alendronate sodium, BP) and nHA favors the uniform dispersion of the latter. Moreover, multiple adhesion ligands based on catechol motifs, BP, and aldehyde groups endow the hydrogels with good tissue adhesion. The hydrogels possess excellent biocompatibility and the introduction of BP and nHA both can effectively promote viability, proliferation, migration, and osteogenesis differentiation of MC3T3-E1 cells. The incorporation of BP groups and HA nanoparticles could also facilitate the angiogenic property of endothelial cells. The nHA/PLGA-Dex DC hydrogels exhibited considerable biocompatibility despite the presence of a certain degree of inflammatory response in the early stage. The successful healing of a rat cranial defect further proves the bone regeneration ability of nHA/PLGA-Dex DC injectable hydrogels. The developed tissue adhesive osteogenic injectable nHA/PLGA-Dex hydrogels show significant potential for bone regeneration application.

Hydroxyapatite-reinforced alginate fibers with bioinspired dually aligned architectures.

Biological materials have excellent mechanical properties due to their organized structures from nano- to macro-scale. Artificial manufacture of materials with anisotropic microstructures still remains challenging. We described a stress-induced method to fabricate anisotropic alginate fibers. Organic-inorganic composite fibers were obtained by incorporating aligned hydroxyapatite (HAP) nanowires into the alginate fiber. Detailed structural characterization revealed the bone-like structure of the HAP-reinforced alginate fibers. Tensile test results showed that the maximum Young's modulus and tensile strength were 4.3 GPa and 153.8 MPa, respectively. A multiscale reinforcing mechanism is proposed after the discussion of the structure-property relationship: highly ordered and compacted nanofibrils aligned along the longitudinal direction at the microscale, and two kinds of alginate gels with different mechanical behaviors at the nanoscale coexisted (acidic alginate gel and calcium-alginate gel). This work validates the effectiveness of the bioinspired fabrication strategy, which inspires further manufacturing and optimization of materials for diverse applications.

Hydrothermal synthesis of hydroxyapatite powders using Response Surface Methodology (RSM).

Hydroxyapatite (HAp)-[Ca10 (PO4)6(OH) 2] has a similar chemical composition to bone material, making it the main mineral supplement in bone-making. Due to its high biocompatibility, hydroxyapatite is widely used in the repair of bone deficiencies and in the production of dental or orthopedic implants. In this research, hydroxyapatite nanopowder was synthesized using a hydrothermal technique. Fourier Transform Infrared Spectroscopy (FTIR) and transmission electron microscopy (TEM) were used to investigate the chemical structure and morphology of the synthesized hydroxyapatite powder. X-ray diffraction (XRD) was used to evaluate the phase analysis of HAp nanopowder. In addition, bioactivity HAp assessment was conducted by scanning electron microscopy (SEM) attached with Energy Dispersive X-Ray Spectroscopy (EDX) analysis. Response Surface Methodology (RSM) with central composite design (CCD) was used in order to determine the optimal conditions for yield, size, and crystallinity. Three independent variables (pH, temperature, and hydrothermal treatment time) were investigated. The yield was observed to increase in alkaline conditions; pH showed the greatest influence on the yield, size, and crystallinity of the synthesized hydroxyapatite, based on Analysis of Variance. The results of bioactivity evaluation are showed high bioactivity due to the formation of apatite on the surface of the synthesized nanopowder.

Efficient antibacterial activity of hydroxyapatite through ROS generation motivated by trace Mn(iii) coupled H vacancies.

Hydroxyapatite (HA) has attracted wide attention for medical application due to its biocompatibility and bioactivity. However, the infection problems of HA remain among the leading reasons for implantation failure. Thus, it is urgent to endow HA biomaterials with antibacterial activity. Herein, the high antibacterial activity was achieved by introducing trace Mn3+ and H vacancy couples in HA through a facile heat-treatment strategy in air. The theoretical results indicated that Mn3+ was preferentially substituted for the Ca(2) site in the HA structure with a charge-compensating H vacancy appearing at the adjacent OH- site. The antibacterial tests showed that Mn-HA possessed antibacterial activity towards both E. coli and S. aureus with trace Mn content at the ppm level, and implied that Mn3+ and centers may play an important role in the antibacterial process. The Mn3+ and couples in Mn-HA, serving as oxidative and reductive centers respectively, could then collectively participate in the CoQ/CoQH2 redox cycling and synergistically facilitate the accumulation of CoQ˙- and ROS radicals. This enhanced ROS production was the main factor to endow Mn-HA with efficient antibacterial activity. Moreover, the in vitro bioactivity assay showed that Mn-HA materials exhibited enhanced osteogenic activity and good biocompatibility. Therefore, this work not only provides a feasible method to control the oxidation state of Mn elements in HA, but also proposes a novel trace Mn3+-doped HA for potential applications in tissue engineering.

Highly porous and elastic aerogel based on ultralong hydroxyapatite nanowires for high-performance bone regeneration and neovascularization.

Hydroxyapatite (HAP) is promising for the clinical treatment of bone defects because of its excellent biocompatibility and osteo-conductivity. However, highly porous HAP scaffolds usually exhibit high brittleness and poor mechanical properties, thus organic constituents are usually added to form composite materials. In this work, a highly porous and elastic aerogel made from ultralong HAP nanowires with ultrahigh porosity (∼98.5%), excellent elasticity and suitable porous structure is prepared as the high-performance scaffold for bone defect repair. The highly porous structure of the as-prepared aerogel is beneficial to bone ingrowth and matter/fluid transfer, and the high elasticity can ensure the structural integrity of the scaffold during bone regeneration. Therefore, the HAP nanowire aerogel scaffold can promote the adhesion, proliferation and migration of rat bone marrow derived mesenchymal stem cells (rBMSCs), and elevate the protein expression of osteogenesis and angiogenesis related genes. The in vivo experimental results demonstrate that the HAP nanowire aerogel scaffold is favorable for the ingrowth of new bone and blood vessels, and thus can greatly accelerate bone regeneration and neovascularization. The as-prepared HAP nanowire aerogel scaffold shows promising potential for biomedical applications such as bone defect repair.

Bioactivity reinforced surface patch bound collagen-pectin hydrogel.

In this report, we discuss the design of a novel collagen/pectin (CP) hybrid composite hydrogel (CPBG) containing in-situ mineralized bioactive glass (BG) particles to simulate an integrative 3D cell environment. Systematic analysis of the CP sol revealed collagen and pectin molecules interacted regardless of both possessing similar net negative charge through the mechanism of surface patch binding interaction. Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) confirmed this associative interaction which resulted in the formation of a hybrid crosslinked network with the BG nanoparticles acting as pseudo crosslink junctions. Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray Analysis (EDAX) and Transmission Electron Microscopy (TEM) results confirmed uniform mineralization of BG particles, and their synergetic interaction with the network. The in-vitro bioactivity tests on CPBG indicated the formation of bone-like hydroxyapatite (Ca10(PO4)6(OH)2) microcrystals on its surface after interaction with simulated body fluid. This hydrogel was loaded with a model antifungal drug amphotericin-B (AmB) and tested against Candida albicans. The AmB release kinetics from the hydrogel followed the Fickian mechanism and showed direct proportionality to gel swelling behavior. Rheological analysis revealed the viscoelastic compatibility of CPBG for the mechanical load bearing applications. Cell viability tests indicated appreciable compatibility of the hydrogel against U2OS and HaCaT cell lines. FDA/PI on the hydrogel portrayed preferential U2OS cell adhesion on hydrophobic hydroxyapatite layer compared to hydrophilic surfaces, thereby promising the regeneration of both soft and hard tissues.

Synthesis and characterization of poly(vinyl alcohol)/chondroitin sulfate composite hydrogels containing strontium-doped hydroxyapatite as promising biomaterials.

Novel poly(vinyl alcohol)/chondroitin sulfate (PVA/CS) composite hydrogels containing hydroxyapatite (HA) or Sr-doped HA (HASr) particles were synthesized by a freeze/thaw method and characterized aiming towards biomedical applications. HA and HASr were synthesized by a wet-precipitation method and added to the composite hydrogels in fractions up to 15 wt%. Physical-chemical characterizations of particles and hydrogels included scanning electron microscopy, energy-dispersive spectroscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, thermogravimetry, porosity, compressive strength/elastic modulus, swelling degree, and cell viability. Particles were irregular in shape and appeared to have narrow size variation. The thermal behavior of composite hydrogels was altered compared to the control (bare) hydrogel. All hydrogels exhibited high porosity. HA/HASr particles reduced total porosity without reducing pore size. The mechanical strength was improved as the fraction of HA or HASr was increased. HASr particles led to a faster water uptake but did not interfere with the total hydrogel swelling capacity. In cell viability essay, increased cell growth (above 120%) was observed in all groups including the control hydrogel, suggesting a bioactive effect. In conclusion, PVA/CS hydrogels containing HA or HASr particles were successfully synthesized and showed promising morphological, mechanical, and swelling properties, which are particularly required for scaffolding.

In Situ Generation of Hydroxyapatite on Biopolymer Particles for Fabrication of Bone Scaffolds Owning Bioactivity.

Hydroxyapatite (HAP) can endow a biopolymer scaffold with good bioactivity and osteoconductive ability, while the interfacial bonding is fairly weak between HAP and biopolymers. In this study, HAP was in situ generated on poly(l-lactic acid) (PLLA) particles, and then they were used to fabricate a scaffold by selective laser sintering. Detailedly, PLLA particles were first functionalized by dopamine oxide polymerization, which introduced abundance active catechol groups on the particle surface, and subsequently, the catechol groups concentrated Ca2+ ions by chelation in a simulated body fluid solution, and then, Ca2+ ions absorbed PO43- ions through electrostatic interactions for in situ nucleation of HAP. The results indicated that HAP was homogeneously generated on the PLLA particle surface, and HAP and PLLA exhibited good interfacial bonding in the HAP/PLLA scaffolds. Meanwhile, the scaffolds displayed excellent bioactivity by inducing apatite precipitation and provided a good environment for human bone mesenchymal stem cell attachment, proliferation, and osteogenic differentiation. More importantly, the ingrowth of blood vessel and the formation of new bone could be stimulated by the scaffolds in vivo, and the bone volume fraction and bone mineral density increased by 44.44 and 41.73% compared with the pure PLLA scaffolds, respectively. Serum biochemical indexes fell within the normal range, which indicated that there was no harmful effect on the normal functioning of the body after implanting the scaffold.

Studies on physiochemical modifications on biologically important hydroxyapatite materials and their characterization for medical applications.

Crystalline Hydroxyapatite (Ca10 (PO4)6(OH)2) non linear optical crystal plays a major role in biomedical applications. Crystalline Hydroxyapatite was synthesized using natural human bone sample by thermal methods. This Hydroxyapatite was characterized by Fourier Transform infra-red spectroscopy (FTIR), scanning electron microscope, UV-Vis spectroscopy, X-ray diffraction and conductivity studies. It was found that compared to bovine bone, the changes to the molecular structure associated with the mineral (carbonate) and the organic content (collagen) was good. The sintering methods showed that the synthesized Hydroxyapatite has remarkable heat stability up to 750 °C. The XRD and FTIR results showed a high purity of the synthesized HA powders, in terms of the electronic transitions, in UV-Vis which require a certain amount of energy it is proportional to the wavelength absorbed and absorption coefficient, the optical band energy of the natural human bone is found to be 4.65 eV and the electrical conductivity of the bone material is 9.11 × 10-6 Ω -1 cm-1. Natural bone Hydroxyapatite gives superior results.

One-step hydrothermal synthesis with in situ milling of biologically relevant hydroxyapatite.

Biologically relevant synthetic hydroxyapatite (HA) has become a much-desired material for use within the medical field with an emphasis on orthopedic applications. However, there are very few sources of sub-micron scale HA powders that are economical. Many current procedures to generate synthetic HA, that is both biological and chemically analogous to naturally occurring HA, tend to involve complicated synthesis procedures that are difficult to simultaneously produce desired stoichiometric ratios and particle diameter. This paper reports the development of a one-step hydrothermal method with in situ ball milling of synthetic HA. That has the potential to be a biological substitute with similar calcium to phosphate stoichiometric ratio and particle diameter of HA found in many natural biologically occulting sources. Parameters affecting particle diameter investigated included varying ball milling media, in situ and ex situ ball milling, and simultaneous agitation. The stoichiometric ratios of the resulting powders indicated that 4-hour hydrothermal reaction time produced materials that are analogous to natural HA, confirmed from spectra acquired via Fourier Transform Infrared spectroscopy (FT-IR). X-ray diffraction and Scanning Electron Microscopy both indicate that the predominant size of primary crystallites is around ~25 nm. Particle size distributions of dried in situ ball-milled HA suggest that primary crystallites exist as aggregates, with aggregate diameters ranging between 1 and 100 µm.

Facile synthesis of biodegradable flower-like hydroxyapatite for drug and gene delivery.

Controlled synthesis of hierarchical hydroxyapatite materials is a hot research topic because of the excellent biocompatibility and bioactivity of the materials. In this study, flower-like hydroxyapatite spheres (FHAPS) were facile synthesized in one pot using Al(OH)3 as a structure-directing agent. The prepared FHAPS comprised nanosheets possessing a uniform diameter of approximately 4 µm. Notably, the FHAPS can be degraded in solutions with a pH of 5.5 for 144 h or incubated with cells for 48 h. In addition, the FHAPS have rough surfaces, which exhibit high loading contents for the anticancer drug doxorubicin (DOX, 9.1%) and siRNA (2.0%). Thus, the FHAPS can effectively deliver DOX into drug-resistant breast cancer cells to exert an excellent killing effect compared with free DOX and transfect siRNA into tumor cells to interfere with the expression of the target protein. Taken together, this work successfully prepared FHAPS via a convenient synthesis route that shows high delivery efficiency for anticancer drugs and siRNA.

Zinc Silicate/Nano-Hydroxyapatite/Collagen Scaffolds Promote Angiogenesis and Bone Regeneration via the p38 MAPK Pathway in Activated Monocytes.

Recent studies show that biomaterials are capable of regulating immune responses to induce a favorable osteogenic microenvironment and promote osteogenesis and angiogenesis. In this study, we investigated the effects of zinc silicate/nanohydroxyapatite/collagen (ZS/HA/Col) scaffolds on bone regeneration and angiogenesis and explored the related mechanism. We demonstrate that 10ZS/HA/Col scaffolds significantly enhanced bone regeneration and angiogenesis in vivo compared with HA/Col scaffolds. ZS/HA/Col scaffolds increased tartrate-resistant acid phosphatase (TRAP)-positive cells, nestin-positive bone marrow stromal cells (BMSCs) and CD31-positive neovessels, and expression of osteogenesis (Bmp-2 and Osterix) and angiogenesis-related (Vegf-α and Cd31) genes increased in nascent bone. ZS/HA/Col scaffolds with 10 wt % ZS activated the p38 signaling pathway in monocytes. The monocytes subsequently differentiated into TRAP+ cells and expressed higher levels of the cytokines SDF-1, TGF-ß1, VEGF-α, and PDGF-BB, which recruited BMSCs and endothelial cells (ECs) to the defect areas. Blocking the p38 pathway in monocytes reduced TRAP+ differentiation and cytokine secretion and resulted in a decrease in BMSC and EC homing and angiogenesis. Overall, these findings demonstrate that 10ZS/HA/Col scaffolds modulate monocytes and, thereby, create a favorable osteogenic microenvironment that promotes BMSC migration and differentiation and vessel formation by activating the p38 signaling pathway.

Synthesis of nanostructured silica/hydroxyapatite hybrid particles containing amphiphilic triblock copolymer for effectively controlling hydration layer structures with cytocompatibility.

We synthesized nanostructured mesoporous silica (MS)/hydroxyapatite (HA) hybrid particles in the presence of amphiphilic poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO20PPO70PEO20) triblock copolymer (P123). The particles exhibited slit-shaped mesostructures and underwent hybridization reaction between the MS and HA phases containing P123. Furthermore, the aggregated form of the particles exhibited dispersion stability in water in the monodispersed state (average particle size: 145 nm and coefficient of variation: 4.3% in the case of the maximum added amount of P123). Then, the structures of the hydration layer and the adsorbed protein on the particles were investigated to understand the effect of the hydration layer structures on the protein secondary structures. The ratio of the bonding water (intermediate and nonfreezing water) to free water increased upon hybridization, and it decreased with increasing P123 concentration. Upon hybridization, the component ratio of the asymmetric O-H stretching vibration between free water molecules decreased, and that of the symmetric O-H stretching vibration of intermediate water molecules increased. With increasing P123 concentration, the asymmetric O-H stretching vibration between free water molecules increased and the symmetric O-H stretching vibration of intermediate water molecules decreased. It was found that the protein native state component ratios of α-helix and ß-sheet increased with increasing symmetric O-H stretching vibration between intermediate water molecules, and they decreased with decreasing asymmetric O-H stretching vibration between free water molecules. Moreover, the cytotoxicity against osteoblasts (MC3T3-E1) was evaluated and the hybrid particles exhibited a high cell density, indicating their bioactivity. On the hybrid particles interacting with P123, the cells were three-dimensionally assembled and uniaxially grown with the culture. Therefore, this is the first successful report of the synthesis of nanostructured MS/HA hybrid particles interacting with P123, and the controlled hydration layer structures on the particle surfaces were found to contribute to the protein secondary structures, promoting cytocompatibility.

Development of surface functionalized hydroxyapatite nanoparticles for enhanced specificity towards tumor cells.

Nanoparticles coupled with targeting moieties have attracted a great deal of attention for cancer therapy since they can facilitate site-specific delivery of drug and significantly limit the side effects of systemic chemotherapy. In this study, our aim is to develop surface functionalized hydroxyapatite nanoparticles, which could provide binding sites for a cancer cell targeting ligand, folic acid (FA) as well as an anticancer drug, doxorubicin hydrochloride (DOX). In order to attain dual functionalities, hydroxyapatite nanoparticles were functionalized with gelatin molecules. Gelatin, being a protein has both carboxyl and amine moieties, which makes it suitable for binding of DOX and FA. FA was chemically conjugated to the nanoparticles through an EDCNHS coupling reaction. The formation of single-phase hydroxyapatite nanostructure was ascertained by X-ray diffraction studies and the presence of organic moieties on the surface of nanoparticles was evident from Fourier transform infrared spectroscopy, thermogravimetric analysis and U.V.-visible spectroscopy. The FA-conjugated nanoparticles (FA-Gel-HANPs) showed high affinity towards DOX and pH-responsive sustained release of drug with higher release rate under acidic pH conditions, desired for cancer therapy. The FA-Gel-HANPs showed negligible cytotoxicity towards different cell lines (HepG2, WEHI-164, KB, WI-26 VA4 and WRL-68). However, DOX loaded nanoparticles (DOX-FA-Gel-HANPs) exhibited significant toxicity towards these cells, which was however highest in folate receptor (FR)-overexpressing, KB cells. These results were correlated with enhanced cellular uptake of DOX-FA-Gel-HANPs in KB cells in comparison to FR-deficient, WRL-68 cells studied by confocal laser scanning microscopy and flow cytometry. Moreover, cell cycle analysis in KB cells, showed higher sub-G1 population, indicating apoptosis as one of the cell death mechanisms. Overall, this study suggests that DOX-FA-Gel-HANPs could serve as a promising tumor-targeted drug delivery system.