Di β glycerophosphate

For the purpose of evaluating how osteogenic differentiation could be influenced by titanium surfaces, cells were seeded at 2 × 10⁴ cells/cm² on these disks. After 72 h of culture, the standard culture medium was replaced with the osteogenic medium (α-MEM, 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL di streptomycin, 100 nM dexamethasone, 10 mM di β-glycerophosphate, all from Sigma-Aldrich, St. Louis, MO, USA). After 3 weeks of induction, immunofluorescence analysis was performed in order to assess the expression of typical differentiation markers, such as RUNX-2 and osteocalcin (OCN). The following primary antibodies were used, at a 1:50 dilution: rabbit anti-RUNX-2, mouse anti-OCN (Abcam, Cambridge, UK).

In order to evaluate the ability of SLA titanium and CERID surfaces to affect osteogenic differentiation, hDPSCs were seeded onto disks (3 × 10³ cells/cm²) and cultured under standard conditions for 72 h. Subsequently, standard culture medium was replaced with osteogenic medium (α-MEM, 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL di streptomycin, 100 nM dexamethasone, 10 mM di β-glycerophosphate, 100µM ascorbic acid, all from Sigma-Aldrich, St. Louis, MO, USA). After 3 weeks of induction, the expression of osteogenic markers, RUNX2, osteopontin (OPN) and osteocalcin (OCN), was assayed through immunofluorescence analyses. Rabbit anti-RUNX2, mouse anti-OPN and mouse anti-OCN (Abcam, Cambridge, UK) primary antibodies were incubated at a 1:50 dilution and revealed with anti-rabbit AlexaFluor546 and anti-mouse Alexa fluor488 secondary antibodies (1:200; Thermo Fisher Scientific). Nuclei were counterstained with DAPI [26 (link)].

In order to evaluate the ability of the BGMS10 surfaces to influence osteogenic differentiation, cells were seeded at approximately 2.5 × 10³ cells/cm² on these disks. After one week of culture, the standard culture medium was replaced with the osteogenic medium (α-MEM, 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL di streptomycin, 100 nM dexamethasone, 10 mM di β-glycerophosphate, all from Sigma-Aldrich, St. Louis, MO, USA). After 3 weeks of induction, the expression of typical differentiation markers, such as RUNX2, osterix (Osx), osteopontin (OPN) and osteocalcin (OCN), was investigated by immunofluorescence analyses as described above. The following primary antibodies were used, at a 1:100 dilution: rabbit anti-RUNX2, mouse anti-OPN, mouse anti-OCN (Abcam, Cambridge, UK) and rabbit anti-Osx (Gene-Tex, San Antonio, TX, USA).