Citrate regulates extracellular matrix mineralization during osteoblast differentiation in vitro.

The extremely high levels of citrate in bone highlight its important role, which must be involved in some essential functional or structural role that is required for the development and maintenance of normal bone. However, biomineralization researches have emphasized the interaction between the citrate and inorganic minerals during crystallization in cell-free systems. It is difficult to obtain a thorough and comprehensive understanding from cell-free experimental conditions and treatment methods. In this study, by proposing an osteoblast mineralization experimental model, we explored the regulation of citrate on bone apatite crystal structure. Our studies show that citrate stabilizes two precursors and then inhibits their transformation into hydroxyapatite. Concomitantly, the smaller size and lower crystallinity mineral deposition emerge during citrate-mediated osteogenic mineralization. These findings may provide a new perspective for the mechanism of osteogenic mineralization and a basis for further understanding of bone metabolism.

Magnesium Calcium Phosphate Cement Incorporating Citrate for Vascularized Bone Regeneration.

The development of bioactive bone cement is still a challenge for vascularized bone regeneration. Citrate participated in multiple biological processes, such as energy metabolism, osteogenesis, and angiogenesis. However, it is difficult to obtain a thorough and comprehensive understanding on osteogenic effects of exogenous citrate from different experimental conditions and treatment methods. In this study, by using a magnesium calcium phosphate cement (MCPC) matrix, we investigated the dual effect of exogenous citrate on osteogenesis and angiogenesis. Our studies show that citrate elevates the osteogenic function of osteoblasts under low doses and the angiogenic function of vascular endothelial cells under a broader dose range. These findings furnish a new strategy for regulating angiogenesis and osteogenic differentiation by administration of citrate in MCPC, driving the development of bioactive bone repair materials.

Citric acid enhances the physical properties, cytocompatibility and osteogenesis of magnesium calcium phosphate cement.

In recent years, the magnesium phosphate cements showed impressive advantages for their setting property, mechanical strength, and resorption rate in laboratory investigation. While it remained a big challenge to develop the magnesium phosphate cements with ideal self-setting properties, sufficient mechanical strength, excellent biocompatibility, and osteoinductivity for clinical application. In our work, we prepared the magnesium calcium phosphate cement (MCPC) using the MgO, KH2P2O4, and Ca(H2PO4)2 particles with the citric acid added. The citric acid was adopted to modify the setting time and compressive strength of the MCPC, which were investigated by the X-ray diffractometer and scanning electron microscopy. The cytocompatibility and osteoinductivity of the modified cements were evaluated by the MC3T3-E1 cells proliferation and morphology, alkaline phosphatase assay, alizarin red staining and western blot assay. The results demonstrated that the citric acid modified MCPC was featured of satisfactory setting time, ideal mechanical strength, good cytocompatibility and osteoinductivity, indicating its potential application for bone regeneration.

Magnesium phosphate based cement with improved setting, strength and cytocompatibility properties by adding Ca(H2PO4)2·H2O and citric acid.

Inorganic phosphate cements have become prevalent as bone filling materials in clinical applications, owing to beneficial properties such as self-setting, biodegradability and osteoconductivity. However, the further development of phosphate cements with higher strength and improved cytocompatibility is expected. In this paper, we reported the preparation of a novel magnesium phosphate based cement (MPBC), which has similar compositions with magnesium phosphate cement (MPC) but Ca(H2PO4)2·H2O and citric acid were additionally added to modulate the performance. The physicochemical and biological properties of MPBC were investigated, the influences of the added Ca(H2PO4)2·H2O and citric acid on the performances of MPBC were analyzed, and the differences of performance between MPBC and MPC were discussed. Experimental results show that the setting time and compressive strength of MPBC were effectively enhanced by the addition of citric acid. In vitro biological degradation indicates that about 15 wt% of MPBC was reduced in 4 weeks. Compared with MPC, MPBC has weaker alkalinity and less dissolution of phosphate, leading to better suitability for cell proliferation and adhesion. These results suggest that as a bone filling material, MPBC shows better performance than MPC in many key indicators and has promising application prospects.

Manipulating Mesenchymal Stem Cells Differentiation Under Sinusoidal Electromagnetic Fields Using Intracellular Superparamagnetic Nanoparticles.

In this study, hollow mesoporous ferrite nanoparticles (HMFNs) were prepared. It showed a spherical morphology with a diameter about 320 nm, with a negatively charged surface, and with a great superparamagnetic property. Negative charge attribute to the free -OH group of the HMFNs shell, which improved nanoparticles hydrophilic and biocompatibility. Superparamagnetic property could avoids particle agglomeration. The particles were shown to be internalized into the bone marrow mesenchymal stem cells (BMSCs) in vitro. We found that the intracellular HMFNs can improve the osteogenic differentiation of BMSCs in the presence of an electromagnetic fields. To determine the optimal intensity of the sinusoidal electromagnetic field (SEMF), the exposure levels of 50 Hz SEMF in the range of 0∼4 mTs (60 min/day) were utilized to investigate its effects on the proliferation and osteogenic differentiation of rat BMSCs. The result showed that the 1 mT and 2 mT SEMF stimulated the BMSCs proliferation significantly. The internalized HMFNs in conjunction with SEMF exposure enhanced the osteogenic differentiation, as evidenced by elevated alkaline phosphatase activity, calcium deposition, and the expression protein levels of the expression profile of osteopontin, osteocalcin and runt-related transcription factor 2. We believe that the electromagnetic fields can manipulate osteogenic differentiation of BMSCs using intracellular superparamagnetic nanoparticles.

Magnetically targeted co-delivery of hydrophilic and hydrophobic drugs with hollow mesoporous ferrite nanoparticles.

A magnetically targeted drug delivery system (DDS) is developed to solve the delivery problem of hydrophobic drugs by using hollow mesoporous ferrite nanoparticles (HMFNs). The HMFNs are synthesized by a one-pot hydrothermal method based on the Ostwald ripening process. The biocompatibility of the synthesized HMFNs was determined by MTT assay, lactate dehydrogenase (LDH) leakage assay and hemolyticity against rabbit red blood cells. Moreover, Prussian blue staining and bio-TEM observations showed that the cell uptake of nanocarriers was in a dose and time-dependent manner, and the nanoparticles accumulate mostly in the cytoplasm. A typical highly hydrophobic anti-tuberculosis drug, rifampin (RFP) was loaded into HMFNs using supercritical carbon dioxide (SC-CO2) impregnation, and the drug loading amount reached as high as 18.25 wt%. In addition, HMFNs could co-encapsulate and co-deliver hydrophobic (RFP) and hydrophilic (isoniazide, INH) drugs simultaneously. The in vitro release tests demonstrated extra sustained co-release profiles of rifampicin and isoniazide from HMFNs. Based on this novel design strategy, the co-delivery of drugs in the same carrier enables a drug delivery system with efficient enhanced chemotherapeutic effect.