Wanyin Zhai

Silicate bioceramics induce angiogenesis during bone regeneration.

The capacity to induce rapid vascular ingrowth during new bone formation is an important feature of biomaterials that are to be used for bone regeneration. Akermanite, a Ca-, Mg- and Si-containing bioceramic, has been demonstrated to be osteoinductive and to promote bone repair. This study further demonstrates the ability of akermanite to promote angiogenesis and investigates the mechanism of this behavior. The akermanite ion extract predominantly caused Si-ion-stimulated proliferation of human aortic endothelial cells. The Si ion in the extract was the most important component for the effect and the most effective concentration was found to be 0.6-2 μg ml(-1). In this range of Si ion concentration, the stimulating effect of the ceramic ion extract was demonstrated by the morphology of cells at the primary, interim and late stages during in vitro angiogenesis using ECMatrix™. The akermanite ion extract up-regulated the expression of genes encoding the receptors of proangiogenic cytokines and also increased the expression level of genes encoding the proangiogenic downstream cytokines, such as nitric oxide synthase and nitric oxide synthesis. Akermanite implanted in rabbit femoral condyle model promoted neovascularization after 8 and 16 weeks of implantation, which further confirmed its stimulation effect on angiogenesis in vivo. These results indicate that akermanite ceramic, an appropriate Si ion concentration source, could induce angiogenesis through increasing gene expression of proangiogenic cytokine receptors and up-regulated downstream signaling. To our knowledge, akermanite ceramic is the first Si-containing ceramic demonstrated to be capable of inducing angiogenesis during bone regeneration.

The stimulation of osteogenic differentiation of human adipose-derived stem cells by ionic products from akermanite dissolution via activation of the ERK pathway.

Our previous study indicates that akermanite, a type of Ca-, Mg-, Si-containing bioceramic, can promote the osteogenic differentiation of hASCs. To elucidate the underlying mechanism, we investigated the effect of the extract from akermanite, on proliferation and osteogenic differentiation of hASCs. The original extract was obtained at 200 mg akermanite/ml LG-DMEM and further diluted with LG-DMEM. The final extracts were denoted as 1/2, 1/4, 1/8, 1/16, and 1/32 extracts based on the concentrations of the original extract. The LDH assay and live/dead stain were used to reveal the cytotoxicity of the different extracts on hASCs, while the DNA assay was carried out to quantitatively evaluate the proliferation of cells after being cultured with the extracts for 1, 3 and 7 days. Flow cytometry for cell cycle analysis was carried out on cells cultured in two media (GM and 1/2 extract) in order to further analyze the effect of the extract on cell proliferation behaviors. Osteogenic differentiation of hASCs cultured in the extracts was detected by ALP expression and calcium deposition, and further confirmed by real-time PCR analysis. It was shown that Ca, Mg and Si ions in the extract could suppress the LDH release and proliferation of hASCs, whereas promote their osteogenic differentiation. Such effects were concentration-dependent with the 1/4 extract (Ca 2.36 mM, Mg 1.11 mM, Si 1.03 mM) being the optimum in promoting the osteogenic differentiation of hASCs. An immediate increase in ERK was observed in cells cultured in the 1/4 extract and such osteogenic differentiation of hASCs promoted by released ions could be blocked by MEK1-specific inhibitor, PD98059. Briefly, Ca, Mg and Si ions extracted from akermanite in the concentrations of 2.36, 1.11, 1.03 mM, respectively, could facilitate the osteogenic differentiation of hASCs via an ERK pathway, and suppress the proliferation of hASCs without significant cytotoxicity.

Stimulatory effects of the ionic products from Ca-Mg-Si bioceramics on both osteogenesis and angiogenesis in vitro.

Ideal biomaterials for bone tissue engineering should have the capability to guide the osteogenic differentiation of mesenchymal stem cells and, at the same time, to stimulate angiogenesis of endothelia cells. In this study it was found that three Ca-Mg-Si-containing bioceramics (bredigite Ca7MgSi4O16, akermanite Ca2MgSi2O7 and diopside CaMgSi2O6) had osteogenic and angiogenic potential. The effects of three silicate ceramics on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and the angiogenesis of human aortic endothelial cells (HAECs) were explored in comparison with β-tricalcium phosphate (β-TCP) bioceramics. The proliferation, alkaline phosphatase (ALPase) activity and bone-related gene expression (COL1, ALPase, OP, BSP and OC) of hBMSCs were significantly enhanced upon stimulation with ionic extracts of these silicate bioceramics. In addition, the results showed that extracts from the three silicate bioceramics also stimulated HAEC proliferation and in vitro angiogenesis with improved NO synthesis and angiogenic gene expression (KDR, FGFR1, ACVRL1 and NOS3). Among the three silicate ceramics bredigite showed the highest osteogenic and angiogenic potential and with the highest extract Si (possibly Si(OH)3O(-)) concentration, while diopside had the lowest osteogenic and angiogenic potential with the lowest extract Si concentration. Furthermore, it was found that the concentration of Si ions in extracts of the three silicate bioceramics was obviously higher than that of β-TCP ceramics, indicating an important role of Si ions in stimulating cell proliferation, osteogenic differentiation and angiogenesis. The results suggest that the silicate-based akermanite and bredigite ceramics might be good scaffold biomaterials for bone tissue engineering applications due to their distinctive dual functions of osteogenesis/angiogenesis stimulation.

Effect of tricalcium silicate on the proliferation and odontogenic differentiation of human dental pulp cells.

INTRODUCTION This study was to investigate the effects of tricalcium silicate (Ca(3)SiO(5)) on proliferation and odontogenic differentiation of human dental pulp cells (hDPCs) in vitro. METHODS The hDPCs were seeded in culture medium with or without Ca(3)SiO(5) extract and calcium hydroxide (Ca(OH)(2)) extract. Proliferation of the hDPCs was measured by methyl-thiazol-tetrazolium (MTT) assay. Odontogenic differentiation of hDPCs was evaluated by real-time polymerase chain reaction by using odontogenic marker genes such as dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP 1), osteocalcin (OC), alkaline phosphatase (ALP), and collagen type I (Col I), which were verified by ALP activity assessment, mineralization assay, and immunocytochemistry staining for dentin sialoprotein (DSP). RESULTS The MTT assay showed that hDPCs cultured with Ca(3)SiO(5) extract proliferated more significantly as compared with Ca(OH)(2) extract. Analysis of odontogenic marker genes indicated that Ca(3)SiO(5) enhanced the expression of those genes. Moreover, the extract of Ca(3)SiO(5) stimulated mineralization and increased ALP and DSP production conspicuously. CONCLUSIONS These results reveal that Ca(3)SiO(5) can induce the proliferation and odontogenic differentiation of hDPCs in vitro and might be a potential candidate for preparation of a new type of Ca(3)SiO(5-)based cement as a pulp-capping agent.

Flower-Like Hierarchically Nanostructured Hydroxyapatite Hollow Spheres: Facile Preparation and Application in Anticancer Drug Cellular Delivery

Flower-like nanostructured hydroxyapatite hollow spheres (NHHS) assembled with nanosheets with a hierarchical morphology are fabricated by a rapid microwave-assisted hydrothermal route. The presence and concentration of block copolymer poly(lactide)-block-poly(ethylene glycol) (PLA-PEG) are important parameters for the formation of the hollow structure. The possible formation mechanism of NHHS is proposed. The NHHS are explored as anticancer drug carriers for cellular delivery of mitoxantrone (MIT). The MIT-loaded NHHS exhibit sustained-drug-release behavior in vitro and the intracellular drug-distribution tests indicate that the MIT loaded in NHHS carriers can enter the cells efficiently. The experiments also show that the NHHS have a good biocompatibility, and therefore, they are promising anticancer drug carriers in cancer chemotherapy.

Hydroxyapatite nanorods/poly(vinyl pyrolidone) composite nanofibers, arrays and three-dimensional fabrics: electrospun preparation and transformation to hydroxyapatite nanostructures.

Electrospinning has been recognized as an efficient technique for fabricating polymer nanofibrous biomaterials. However, the study of electrospun inorganic biomaterials with well-designed three-dimensional (3-D) structures is still limited and little reported. In this study hydroxyapatite (HAp) nanorods with an average diameter of approximately 7 nm and length of approximately 27 nm were synthesized through a simple precipitation method and used for the fabrication of inorganic/organic [poly(vinyl pyrolidone) (PVP)] composite nanofibers by electrospinning in ethanol solution. 3-D fabrics and aligned nanofiber arrays of the HAp nanorods/PVP composite were obtained as precursors. Thereafter, 3-D single phase HAp fabrics, tubular structures and aligned nanofiber arrays were obtained after thermal treatment of the corresponding composite precursors. Cytotoxicity experiments indicated that the HAp fabric scaffold had good biocompatibility. In vitro experiments showed that mesenchymal stem cells could attach to the HAp fabric scaffold after culture for 24h.

Degradation of hollow mesoporous silica nanoparticles in human umbilical vein endothelial cells

Hollow mesoporous silica nanoparticles (HMSNs) are considered a potential drug delivery system owing to their recognized advantages in drug loading and releasing. However, whether HMSN could be degraded inside the cells remains unknown. In this study, based on the observations by transmission electron microscopy, fluorescence staining, enzymatic proteolysis, and inductively coupled plasma atomic emission spectroscopy, HMSNs were proved to be degradable in human umbilical vein endothelial cells. The degradation first took place in cytoplasm and lysosomes, and secondly in lysosomes only. The Si content in culture medium increased as the time increases, suggesting that the degradation product inside the cells could be excreted into the culture medium. The degrading rate is fast in the first 2 days and slow after 2 days. The present results provided a clue to further research on the metabolic way and cytotoxicity of silica nanoparticles.

Hierarchically structured nanocrystalline hydroxyapatite assembled hollow fibers as a promising protein delivery system

Nanocrystalline hydroxyapatite assembled hollow fibers (NHAHF) in the membrane form were fabricated by combining the electrospinning technique and the hydrothermal method. This novel hierarchical tubular structure of hydroxyapatite exhibited excellent protein loading capacity and long-term sustained release property.

Quercetin-crosslinked porcine heart valve matrix: mechanical properties, stability, anticalcification and cytocompatibility.

Bioprosthetic heart valves, prepared by glutaraldehyde (GA) crosslinking, have some limitations due to poor durability, calcification and immunogenic reactions. The aim of this study was to evaluate the crosslinking effect of a natural product, quercetin, on decellularized porcine heart valve extracellular matrix (ECM). After crosslinking, the mechanical properties, stability, anticalcification and cytocompatibility were examined. The results showed that the tensile strength of quercetin-crosslinked ECM was higher than that of GA-crosslinked ECM. After crosslinking with quercetin, the thermal denaturation temperature of ECM was clearly increased. Quercetin-crosslinked ECM could be stored in D-Hanks solution for at least 30 days without any loss of ultimate tensile strength and elasticity. After soaking in D-Hanks solution for 36 days, there was only 11.55% non-crosslinked excess quercetin released and no further release thereafter. Cell culture study shows that no inhibition on proliferation of vascular endothelial cells occurred when the quercetin concentration was lower than 1microg ml(-1). This non-cytotoxic concentration was 100 times higher than that of GA. The resistibility of quercetin-crosslinked ECM to in vitro enzymatic hydrolysis was comparable to that of GA-crosslinked ECM. An in vitro anticalcification experiment showed that quercetin crosslinking could protect ECM from deposition of minerals in simulated body fluid. The present study demonstrated that quercetin can crosslink porcine heart valve ECM effectively, which suggests that quercetin might be a new crosslinking reagent for the preparation of bioprosthetic heart valve xenografts and scaffolds for heart valve tissue engineering.

Preparation and in vitro evaluation of plasma-sprayed Mg2SiO4 coating on titanium alloy

In this paper, chemically synthesized Mg(2)SiO(4) (MS) powder was plasma-sprayed onto a titanium alloy substrate to evaluate its application potentials in biomedicine. The phase composition and surface morphology of the MS coating were analyzed. Results showed that the MS coating was composed mainly of Mg(2)SiO(4) phase, with a small amount of MgO and glass phases. Mechanical testing showed that the coating exhibited good adhesion strength to the substrate due to the close thermal expansion coefficient between the MS ceramic and the titanium alloy substrate. The measured bonding strength was as high as 41.5+/-5.3MPa, which is much higher than the traditional HA coating. In vitro cytocompatibility evaluation of the MS coating was performed using canine bone marrow stem cells (MSCs). The MSCs exhibited good adhesion, proliferation and differentiation behavior on the MS coating surface, which can be explained by the high protein adsorption capability of the MS coating, as well as the stimulatory effects of Mg and Si ions released from the coating. The proliferation rate of the MSCs on MS coating was very close to that on the hydroxylapatite (HA) coating. Alkaline phosphatase (ALP) activity analysis demonstrated that the ALP level of the MSCs on the MS coating remained high even after 21days, implying that the surface characteristics of the coating are beneficial for the differentiation of MSCs. In summary, our results suggest that MS coating might be a new approach to prepare bone implants.