Enhanced growth and osteogenic differentiation of human osteoblast-like cells on boron-doped nanocrystalline diamond thin films.

Intrinsic nanocrystalline diamond (NCD) films have been proven to be promising substrates for the adhesion, growth and osteogenic differentiation of bone-derived cells. To understand the role of various degrees of doping (semiconducting to metallic-like), the NCD films were deposited on silicon substrates by a microwave plasma-enhanced CVD process and their boron doping was achieved by adding trimethylboron to the CH(4):H(2) gas mixture, the B∶C ratio was 133, 1000 and 6700 ppm. The room temperature electrical resistivity of the films decreased from >10 MΩ (undoped films) to 55 kΩ, 0.6 kΩ, and 0.3 kΩ (doped films with 133, 1000 and 6700 ppm of B, respectively). The increase in the number of human osteoblast-like MG 63 cells in 7-day-old cultures on NCD films was most apparent on the NCD films doped with 133 and 1000 ppm of B (153,000 ± 14,000 and 152,000 ± 10,000 cells/cm(2), respectively, compared to 113,000 ± 10,000 cells/cm(2) on undoped NCD films). As measured by ELISA per mg of total protein, the cells on NCD with 133 and 1000 ppm of B also contained the highest concentrations of collagen I and alkaline phosphatase, respectively. On the NCD films with 6700 ppm of B, the cells contained the highest concentration of focal adhesion protein vinculin, and the highest amount of collagen I was adsorbed. The concentration of osteocalcin also increased with increasing level of B doping. The cell viability on all tested NCD films was almost 100%. Measurements of the concentration of ICAM-1, i.e. an immunoglobuline adhesion molecule binding inflammatory cells, suggested that the cells on the NCD films did not undergo significant immune activation. Thus, the potential of NCD films for bone tissue regeneration can be further enhanced and tailored by B doping and that B doping up to metallic-like levels is not detrimental for cells.

Nanodiamond as promising material for bone tissue engineering.

The adhesion, growth and differentiation of human osteoblast-like MG 63 cells were investigated in cultures grown on nanostructured nanocrystalline diamond (NCD) films with either low surface roughness (rms of 8.2 nm) or hierarchically organized surfaces made of low roughness NCD films deposited on Si surfaces with the original microroughness (rms of 301.0 nm and 7.6 nm, respectively). The NCD films were grown using a microwave plasma-enhanced CVD method in an ellipsoidal cavity reactor. The films were treated in oxygen plasma to enhance the hydrophilic character of the diamond surface (water drop contact angle approx. 20 degrees). The samples were then sterilized by 70% ethanol, inserted into 12-well polystyrene multidishes (diameter 2.2 cm), seeded with human osteoblast-like MG 63 cells (40,000 cells/dish, 10,530 cells/cm2) and incubated in 2 ml of DMEM medium with 10% of fetal bovine serum. On day 3 after seeding, the cell numbers were significantly higher on the nanostructured NCD films (72,020 +/- 6540 cells/cm2) and also on the hierarchically micro- and nanostructured films (60200 +/- 6420 cells/cm2) than on the control polystyrene culture dish (40750 +/- 2,530 cells/cm2). The cells on hierarchically micro- and nanostructured diamond substrates also adhered over a significantly larger area (3730 +/- 180 microm2 compared to 2740 +/- 130 microm2 on polystyrene). The cell viability, measured by a LIVE/DEAD viability/cytotoxicity kit, reached 98% to 100% on both types of NCD films. The XTT test showed that the cells on both nanodiamond layers had significantly higher metabolic activity than those on the control polystyrene dish (approx. 2 to 3 times). Immunofluorescence staining of the cells on both NCD films revealed talin-containing focal adhesion plaques and beta-actin filaments, well apparent particularly at the cell periphery, as well as the presence of considerable amounts of osteocalcin, i.e., a marker of osteogenic cell differentiation. These results suggest that nanocrystalline diamond films give good support for adhesion, growth and differentiation of osteogenic cells and could be used for surface modification of bone implants in order to improve their integration with the surrounding bone tissue.

The influence of pore size on colonization of poly(L-lactide-glycolide) scaffolds with human osteoblast-like MG 63 cells in vitro.

A degradable copolymer of L-lactide and glycolide (PLG) was synthesized by ring opening polymerization using zirconium acetylacetonate [Zr(acac)(4)] as a biocompatible initiator. The structure of the copolymer was studied by nuclear magnetic resonance spectroscopy (NMR) and gel permeation chromatography (GPC). Porous scaffolds of defined microstructure were prepared by solvent casting/salt particulate leaching, which resulted in the creation of three types of scaffolds with the same porosity (87%+/-1%) but with different diameters of the pores (600, 200 and 40 microm) and degree of interconnectivity. The potential of the scaffolds for cell colonization was tested in a conventional static cell culture system using human osteoblast-like MG 63 cells. As revealed by conventional fluorescence and confocal microscopy on days 5 and 7 after seeding, the cells on the scaffolds of large or medium pore size infiltrated the inside part of the material, whereas on the scaffolds of small pore size, the cells were retained on the material surface. On day 7 after seeding, the highest number of cells was found on the scaffolds of the largest pore size (more than 120,000 cells per sample of the diameter 15 mm and thickness 2 mm), whereas on the scaffolds with medium and smallest pore diameter, the number of cells was almost three times lower and similar for both pore sizes. These results corresponded well with the incorporation of bromodeoxyuridine into newly synthesized DNA, which was significantly higher in cells on scaffolds of the largest pore size than on the material with medium and smallest pore diameter. As indicated by the MTT test, the mitochondrial activity in cells on scaffolds with medium pore size was similar to that on the material with the highest pore size, and significantly higher than on scaffolds of the smallest pore diameter. These results suggest that PLG scaffolds with the largest pore diameter (600 microm) and better pore interconnectivity are the most suitable for colonization with osteogenic cells.

Dual activity of triterpenoids: apoptotic versus antidifferentiation effects.

Triterpenoids are natural, biologically active compounds extracted from many plants. They possess antiinflammatory, anticancer, and antioxidant properties. In the report presented, antiproliferative effects and leukemia cell growth and apoptosis modulating activities of ursolic acid (UA) and oleanolic acid (OA) were investigated. Both triterpenoids are inhibitors of leukemia cell growth and inductors of apoptosis. However, when applied in combination with anthracycline antitumor antibiotic doxorubicin (Dox), UA and OA diversely modulate therapeutic efficacy of Dox, due to different antioxidant activities. Compare to OA showing synergism/additive effect with Dox, UA (stronger antioxidant) acts antagonistically and reduces leukemia cell growth inhibiting and differentiation effects induced by Dox. In conclusion, these findings suggest that although triterpenoids UA and OA can induce apoptosis, their antioxidant activities can interfere with the therapeutic effect of antitumor antibiotic Dox which mechanism of action is attributed to the production of reactive oxygen species.