Characterization of growth and osteogenic differentiation of rabbit bone marrow stromal cells.
BACKGROUND
The rabbit is recognized as an excellent model to study the repair of bony defects with tissue engineered constructs. However, the use of rabbit bone marrow stromal cells (RBMSCs) has been limited despite the proven benefits of autologous BMSCs in the formation of bone. The purpose of this study was to characterize the growth and differentiation pattern of RBMSCs and their response to growth factors.


METHODS
BMSCs were isolated from New Zealand White rabbits and cultured. Serial cell counts of parallel cultures were taken daily to determine cell growth. Response of RBMSCs to varying doses of recombinant human BMP-2 (rhBMP-2) and their time course was gauged by alkaline phosphatase (ALP) activity. The osteoblastic differentiation potential of RBMSCs in response to rhBMP-2 treatment was determined by evaluating the expression pattern of various genes involved with osteogensis using northern analysis. Von Kossa staining was performed to determine the effect of rhBMP-2 on the mineralization capabilities of RBMSCs.


RESULTS
The growth rate of RBMSCs severely declined after first passage and this rate was further suppressed by TGF-beta1. The optimal dose response of rhBMP-2 was determined to be 50 ng/ml and its time course displayed increasing alkaline phosphatase activity over time. Two osteogenic markers, collagen I and osteopontin, were up regulated by rhBMP-2 treatment. Finally, the mineralization capability of RBMSCs was determined to be enhanced by rhBMP-2 treatment.


CONCLUSIONS
Our work indicates that RBMSCs possess strong osteogenic potential and can be successfully applied toward bone tissue engineering in a rabbit model.
Roostaeian
Jason
J
Plastic Surgery Section, VA Greater LA Healthcare System, Los Angeles, California 90073, USA.
Carlsen
Brian
B
Simhaee
David
D
Jarrahy
Reza
R
Huang
Weibiao
W
Ishida
Kenji
K
Rudkin
George H
GH
Yamaguchi
Dean T
DT
Miller
Timothy A
TA
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002113:Calcification, Physiologic / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000187:drug effects; D010012:Osteogenesis / Q000187:drug effects* / Q000502:physiology; D015676:Osteonectin / Q000235:genetics; D053495:Osteopontin; D011817:Rabbits; D012795:Sialoglycoproteins / Q000235:genetics; D017154:Stromal Cells / Q000166:cytology*; D023822:Tissue Engineering / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D015676:Osteonectin; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D053495:Osteopontin