Osteogenic differentiation is inhibited and angiogenic expression is enhanced in MC3T3-E1 cells cultured on three-dimensional scaffolds.
Osteogenic differentiation of osteoprogenitor cells in three-dimensional (3D) in vitro culture remains poorly understood. Using quantitative real-time RT-PCR techniques, we examined mRNA expression of alkaline phosphatase, osteocalcin, and vascular endothelial growth factor (VEGF) in murine preosteoblastic MC3T3-E1 cells cultured for 48 h and 14 days on conventional two-dimensional (2D) poly(l-lactide-co-glycolide) (PLGA) films and 3D PLGA scaffolds. Differences in VEGF secretion and function between 2D and 3D culture systems were examined using Western blots and an in vitro Matrigel-based angiogenesis assay. Expression of both alkaline phosphatase and osteocalcin in cells cultured on 3D scaffolds was significantly downregulated relative to 2D controls in 48 h and 14 day cultures. In contrast, elevated levels of VEGF expression in 3D culture were noted at every time point in short- and long-term culture. VEGF protein secretion in 3D cultures was triple the amount of secretion observed in 2D controls. Conditioned medium from 3D cultures induced an enhanced level of angiogenic activity, as evidenced by increases in branch points observed in in vitro angiogenesis assays. These results collectively indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate when cultured on 3D PLGA scaffolds and that VEGF is preferentially expressed by these cells when they are cultured in three dimensions.
Plastic Surgery Section, Veterans Administration Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA.
Reza
R
Jarrahy
Weibiao
W
Huang
George H
GH
Rudkin
Jane M
JM
Lee
Kenji
K
Ishida
Micah D
MD
Berry
Modar
M
Sukkarieh
Benjamin M
BM
Wu
Dean T
DT
Yamaguchi
Timothy A
TA
Miller
D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D019344:Lactic Acid; D051379:Mice; D018919:Neovascularization, Physiologic / Q000502:physiology*; D015675:Osteocalcin / Q000096:biosynthesis; D010011:Osteocytes / Q000166:cytology*; D011100:Polyglycolic Acid; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011108:Polymers; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis
D011108:Polymers; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D015675:Osteocalcin; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D000469:Alkaline Phosphatase