The role of phospholipase D in osteoblast response to titanium surface microstructure.
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-D(3) [24R,25(OH)(2)D(3)]. 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)(2)D(3) 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)(2)D(3) 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.
Fang
Mimi
M
Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA.
Olivares-Navarrete
Rene
R
Wieland
Marco
M
Cochran
David L
DL
Boyan
Barbara D
BD
Schwartz
Zvi
Z
D001483:Base Sequence; D002460:Cell Line; D002999:Clone Cells; D004789:Enzyme Activation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000187:drug effects* / Q000201:enzymology*; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D013499:Surface Properties / Q000187:drug effects; D014025:Titanium / Q000737:chemistry* / Q000494:pharmacology*; D014807:Vitamin D / Q000031:analogs & derivatives / Q000378:metabolism
D007527:Isoenzymes; D034741:RNA, Small Interfering; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D014025:Titanium; D011493:Protein Kinase C; D010739:Phospholipase D
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