Human adipose-derived stromal cells respond to and elaborate bone morphogenetic protein-2 during in vitro osteogenic differentiation.
BACKGROUND
Interest in the potential application of adipose-derived stromal cells in cell-mediated tissue engineering of bone and other mesenchymal-derived tissues is growing. This study aimed to investigate the hypothesis that human adipose-derived stromal cells respond to and elaborate bone morphogenetic protein (BMP) 2, which could represent an important target of molecular manipulation to enhance the osteogenic potential of human adipose-derived stromal cells.


METHODS
Human adipose-derived stromal cells were differentiated for 10 days toward the osteogenic lineage in osteogenic differentiation media alone or supplemented with recombinant human BMP2 (rhBMP2). Alizarin red staining was quantified by spectrophotometry. Gene expression analyses were performed using quantitative real-time polymerase chain reaction. BMP2 levels in conditioned media were titered by enzyme-linked immunosorbent assay daily during osteogenic differentiation. Human adipose-derived stromal cells were cultured in complete or partially (50 percent) changed osteogenic differentiation media, or unchanged osteogenic differentiation media, to assay for pro-osteogenic secreted factors. In addition, human adipose-derived stromal cells were cultured in osteogenic differentiation media supplemented with BMP2/BMP4-neutralizing antibody.


RESULTS
Exogenous rhBMP2 significantly augmented the in vitro osteogenic potential of human adipose-derived stromal cells in a dose-dependent fashion, and significantly increased transcript levels of RUNX2 and osteocalcin. BMP2, BMP4, BMPR1B, and SMAD1/5 expression was significantly increased during differentiation. Enzyme-linked immunosorbent assay demonstrated significantly increased BMP2 elaboration during differentiation. Culture in conditioned osteogenic differentiation media led to significantly increased matrix mineralization. Mineralization was significantly decreased when osteogenic differentiation media was supplemented with a BMP2/BMP4-neutralizing antibody.


CONCLUSIONS
These data strongly support that BMP signaling is dynamic and important during normal in vitro osteogenic differentiation of human adipose-derived stromal cells. Thus, BMP2 may be used to enhance the osteogenic differentiation of human adipose-derived stromal cells for bone tissue engineering. Future studies will examine the effect of rhBMP2 on osteogenic differentiation of human adipose-derived stromal cells in vivo.
Panetta
Nicholas J
NJ
Stanford, Calif. From the Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine.
Gupta
Deepak M
DM
Lee
Jacqueline K
JK
Wan
Derrick C
DC
Commons
George W
GW
Longaker
Michael T
MT
D000273:Adipose Tissue / Q000166:cytology*; D000293:Adolescent; D000328:Adult; D000368:Aged; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D055415:Bone Morphogenetic Protein 4 / Q000235:genetics; D052005:Bone Morphogenetic Protein Receptors, Type I / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics; D017077:Culture Media, Conditioned / Q000494:pharmacology; D005260:Female; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D008297:Male; D008875:Middle Aged; D015675:Osteocalcin / Q000235:genetics; D010011:Osteocytes / Q000166:cytology*; D015398:Signal Transduction / Q000187:drug effects; D051898:Smad1 Protein / Q000235:genetics; D051902:Smad5 Protein / Q000235:genetics; D017154:Stromal Cells / Q000166:cytology* / Q000187:drug effects*; D023822:Tissue Engineering / Q000379:methods*; D055815:Young Adult
C525718:BMP2 protein, human; C525809:BMP4 protein, human; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D050659:Core Binding Factor Alpha 1 Subunit; D017077:Culture Media, Conditioned; C493709:RUNX2 protein, human; C497399:SMAD1 protein, human; C497797:SMAD5 protein, human; D051898:Smad1 Protein; D051902:Smad5 Protein; D015675:Osteocalcin; C498214:BMPR1B protein, human; D052005:Bone Morphogenetic Protein Receptors, Type I