A static force scan was performed using a constantly increasing
force (200 mN/min) until the strip (PTFE only n = 2, titanium coated PTFE n = 3, titanium coated PTFE + purmorphamine n = 3) was pulled out of the bone (breaking point) on which point the required force was a quantification for the integration. The hedgehog pathway works over 2 transmembranic proteins; patched (Ptch) and smoothened (Smo), where Smo is activating the Gli protein function and transcription which will further regulate the transcription of proteins important in selleck kinase inhibitor bone formation like Wnt. In the inactive state, Smo is inhibited by Ptch. The sonic hedgehog protein, during bone formation in the developmental stage produced by chondrocytes, will stop this inhibition
and start bone formation (Fig. 1a). Purmorphamine works by directly activating the Smo transmembrane protein regardless whether Ptch is inhibiting Smo or not. This activation was analyzed through the expression of the bone marker Bsp. Q-PCR dCt values using GapdH as an internal control: in negative medium (control): 1w: 14.17, 2W: 13.28; in positive medium: 1w: 13.53, 2W: 10.67; adding dexamethasone to positive medium: 1w: 12.14, 2W: 8.00; using BMP-6: 1w: 11.24, 2W: 8.14; using purmorphamine: 1w: 11.29, 2W: 7.21; using both purmorphamine and BMP-6: 1w: 8.51, 2W: 4.10. Thereby Q-PCR-data STA-9090 purchase showed that the administration of 2 μM purmorphamine had similar effect on the expression of Bsp as both dexamethasone and BMP-6. The upregulation was greater than when positive medium (DMEM + 10%FCS + p/s + Asc + ß-glycerphosphate) was used without extra agonists. This activation by very purmorphamine had an additive effect compared to BMP-6 stimulation as the addition of both simultaneously showed a higher upregulation than each on their own ( Fig. 1b). This shows that purmorphamine is a small
molecule (= non-protein molecule) that can activate the hedgehog pathway and thereby stimulate bone formation. The strong Raman peak at 960 cm− 1, (PO stretch) in the spectrum of pure hydroxyapatite (dark blue spectrum, Fig. 2a) was clearly observed in the Raman spectrum of the CaP coated plastic disc (light blue spectrum, Fig. 2a), but not in the spectrum of the plastic disc without CaP (green spectrum, Fig. 2a). Almost all other peaks from the CaP coated plastic disc were coincident with and therefore attributed to Thermanox® plastic peaks. Only a shoulder-peak around 1065 cm− 1 was not identifiable. This provides strong evidence that the biomimetically precipitated CaP is primarily hydroxyapatite. Further analysis would be required to confirm purity but for our purpose as an agonist delivery mechanism the verification of the CaP coating is sufficient (Fig. 2a). A Raman spectrum of a coated disc with purmorphamine added did not show any detectable differences compared to the spectrum of the coated disc without purmorphamine.