Whether the adhered small molecule kept its activity was analyzed with a dual luciferase cell-based bioassay. As the Light 2 cells are made to express firefly luciferase when the Gli-inducible promoter selleck chemicals is upregulated, the stimulation of the hedgehog pathway can be calculated by measuring this firefly luciferase luminescence; the constitutive Renilla luciferase is a measure for the number of cells (Figs. 2b and c show the cell attachment and spreading onto the CaP coating). The ratio of the two gives the Gli expression per cell, a quantification of the bio-activity of the adhered Pur.

In Fig. 2d, it is shown that the cells growing on the CaP coated discs with Pur expressed more luciferase or Gli compared to those where no CaP was adhered. Soaking the Pur CaP discs in medium once or twice for 24 h showed that not all of the agonist molecules were released immediately but that there was a gradual release up to 2 days and after soaking for 2 days the Gli expression was still being upregulated.

The size of the HA-porous beads (+/− 30–150 μm) could be adjusted by increasing the speed of the stirring, the faster the stirring; the smaller the beads. A uniformity of the bead-size was not a necessity as beads with a similar size (+/− 50 μm) could be selected afterwards. The CaP beads with an appropriate PF-562271 size could easily be pushed in the defect with the tip of a 27 gage needle. The chick femurs with the implants inserted were overgrown by vessels from the chicken CAM and could thereby remain vital. The femurs had even grown in thickness during the 7 days they were on the CAM incubated at

37 °C. During the sectioning of the middle part of the bone care was taken to ensure that the bones were not over-decalcified and the site of implant and the CaP beads could be retrieved. The toluidine blue stained sections showed the difference in bone growth between the controls and the femurs Thymidylate synthase where beads with agonists had been implanted (Figs. 3a and b). To quantify the bone growth the size of the overall bone area, and the trabecular bone area were measured and the proportion of trabecular bone area to the bone marrow was compared between the different samples, the average 68.19 +/− 7.13% trabecular bone to overall bone area of the test (Pur) samples was significantly higher than the 48.25 +/− 6.52% of the control samples, showing the in vivo effect of the adhered small molecule in and on the implanted CaP beads. The only selection from the bone marrow cell population was made by removing the non-sticky cells when the medium was refreshed, making it a rather stem-cell rich cell-mixture, similar to the bone marrow. But a significant increase in alkaline phosphatase activity (this is a marker for osteodifferentiation of the cells) was seen when BMP-6 (31.44 +/− 4.63) or Pur (31.27 +/− 5.86) was added to the positive medium (7.37 +/− 2.07) as shown by the PNPP-spectroscopy results in Fig.