The role of phospholipase D in osteoblast response to titanium surface microstructure

Abstract Biomaterial surface properties such as microtopography and energy can change cellular responses at the cell‐implant interface. Phospholipase D (PLD) is required for the differentiation of osteoblast‐like MG63 cells on machined and grit‐blasted titanium surfaces. Here, we determined if PLD is also required on microstructured/high‐energy substrates and the mechanism involved. shRNAs for human PLD1 and PLD2 were used to silence MG63 cells. Wild‐type and PLD1 or PLD1/2 silenced cells were cultured on smooth‐pretreatment surfaces (PT); grit‐blasted, acid‐etched surfaces (SLA); and SLA surfaces modified to have higher surface energy (modSLA). PLD was inhibited with ethanol or activated with 24,25‐dihydroxyvitamin‐D₃ [24R,25(OH)₂D₃]. As surface roughness/energy increased, PLD mRNA and activity increased, cell number decreased, osteocalcin and osteoprotegerin increased, and protein kinase C (PKC) and alkaline phosphatase specific activities increased. Ethanol inhibited PLD and reduced surface effects on these parameters. There was no effect on these parameters after knockdown of PLD1, but PLD1/2 double knockdown had effects comparableto PLD inhibition. 24R,25(OH)₂D₃ increased PLD activity and the production of osteocalcin and osteoprotegerin, but decreased cell number on the rough/high‐energy surfaces. These results confirm that surface roughness/energy‐induced PLD activity is required for osteoblast differentiation and that PLD2 is the main isoform involved in this pathway. PLD is activated by 24R,25(OH)₂D₃ in a surface‐dependent manner and inhibition of PLD reduces the effects of surface microstructure/energy on PKC, suggesting that PLD mediates the stimulatory effect of microstructured/high‐energy surfaces via PKC‐dependent signaling. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010