Chitosan scaffolds reinforced by beta-tricalcium phosphate (beta-TCP) and calcium phosphate invert glass were fabricated with a low-cost, bioclean freeze-drying technique via thermally induced phase separation. The microstructure, mechanical performance, biodegradation, and bioactivity of the scaffolds were studied. The composite scaffolds were macroporous, and the pore structures of the scaffolds with beta-TCP and the glass appeared very different. Both the compressive modulus and yield strength of the scaffolds were greatly improved, and reinforced microstructures were achieved. The bioactivity tests showed a continuous decrease in both Ca and P concentrations of a simulated body fluid (SBF) after the scaffolds with beta-TCP were immersed in the SBF for more than 20 h, which suggests that an apatite layer might be formed on the scaffolds. However, the same was not observed for the pure chitosan scaffolds or the scaffolds incorporated with the glass. This was further confirmed by micrographs from scanning electron microscopy. This study suggests that the desirable pore structure, biodegradation rate, and bioactivity of the composite scaffolds might be achieved through controlling the ratio of chitosan and calcium phosphates or beta-TCP and the glass.

Macroporous calcium phosphate-chitosan composite scaffolds were fabricated and evaluated for use in bone tissue engineering. Human osteoblast-like MG63 cells were cultured on the composite scaffolds, and their response to the materials was studied. Cell morphology, total protein content, and expression of classic markers for osteoblast differentiation were characterized. MG63 cells on the hydroxyapatite scaffolds nesting chitosan sponges (HC1) showed significantly higher alkaline phosphatase (ALP) level and osteocalcin (OC) production during the 11-day culture period, compared with the control culture on tissue culture plates. Cells on the chitosan scaffolds incorporated with hydroxyapatite powders (HC2) exhibited lower ALP activity during the 11-day culture period and OC secretion during the first 7 days, in comparison with that on HC1. The addition of calcium phosphate glass as in HC3 scaffolds increased the ALP and OC levels of MG63 cells. Our study indicated that the hydroxyapatite-matrix composite scaffolds might enhance the phenotype expression of MG63 cells, in comparison with chitosan-matrix scaffolds. Soluble calcium phosphate glasses should be added to the scaffolds to prevent chitosan from fast degradation that may affect the differentiation of osteoblast cells.

BackgroundThe prognosis for patients with hepatocellular carcinoma (HCC) is poor, and the mechanisms underlying the development of HCC remain unclear. Notch1 and Notch3 may be involved in malignant transformation, although their roles remain unknown.Materials and MethodsHCC tissues were stained with anti-Notch1 or -Notch3 antibody. The migration and invasion capacities of the cells were measured with transwell cell culture chambers. RT-PCR was used to measure the expression of Notch1 and Notch3 mRNA. Additionally, western blot analysis was used to assess the protein expression of Notch1, Notch3, CD44v6, E-cadherin, matrix metalloproteinase-2 (MMP-2), MMP-9, and urokinase-type plasminogen activator (uPA). RNA interference was used to down-regulate the expression of Notch1 and Notch3. Cell viability was assessed using MTT.ResultsBased on immunohistochemistry, high Notch1 expression was correlated with tumor size, tumor grade, metastasis, venous invasion and AJCC TNM stage. High Notch3 expression was only strongly correlated with metastasis, venous invasion and satellite lesions. Kaplan-Meier curves demonstrated that patients with high Notch1 or Notch3 expression were at a significantly increased risk for shortened survival time. In vitro, the down-regulation of Notch1 decreased the migration and invasion capacities of HCC cells by regulating CD44v6, E-cadherin, MMP-2, MMP-9, and uPA via the COX-2 and ERK1/2 pathways. Down-regulation of Notch3 only decreased the invasion capacity of HCC cells by regulating MMP-2 and MMP-9 via the ERK1/2 pathway.ConclusionsBased on the migration and invasion of HCC, we hypothesize that targeting Notch1 may be more useful than Notch3 for designing novel preventive and therapeutic strategies for HCC in the near future.

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