Modulation of osteogenic potential by recombinant human bone morphogenic protein-2 in human periodontal ligament cells: effect of serum, culture medium, and osteoinductive medium.
OBJECTIVE
Bone morphogenic proteins are known, in animal models, to promote many developmental processes, including osteogenesis. Clinical trials are currently underway to evaluate the potential of bone morphogenic proteins to promote bone and periodontal regeneration in humans. The aim of this study was to establish an optimal cell culture condition for using to study the biological effects of recombinant human bone morphogenic protein-2 on periodontal ligament cells.


METHODS
The roles of serum concentration, types of culture medium (alpha-modified essential medium or Dulbecco's modified Eagle's medium), the presence of osteoinductive medium (including dexamethasone, ascorbic acid and beta-glycerophosphate), and timing of addition of the osteoinductive medium and recombinant human bone morphogenic protein-2, on the expression of alkaline phosphatase were investigated in cultured periodontal ligament cells. Cytochemical stainings and biological assay of alkaline phosphatase were also demonstrated.


RESULTS
Our results suggested that an increased concentration of serum might mask the effect of recombinant human bone morphogenic protein-2 on the expression of alkaline phosphatase in periodontal ligament cells. alpha-Modified essential medium was found to induce a stronger cytochemical staining of the alkaline phosphatase than Dulbecco's modified Eagle's medium under similar culture conditions. Pre-incubation of cells with osteoinductive medium before the addition of various concentrations of recombinant human bone morphogenic protein-2 enhanced greater alkaline phosphatase expression than the simultaneous presence of both osteoinductive medium and recombinant human bone morphogenic protein-2.


CONCLUSIONS
The findings of this study suggest that the effect of recombinant human bone morphogenic protein-2 on periodontal ligament cells could be efficiently investigated after the proper selection of culture variables and temporal sequence of adding bioactive factors. The optimal culture condition identified in this study might be useful in further studies to elucidate the regulatory mechanism of periodontal ligament cells in periodontal regeneration after stimulation with recombinant human bone morphogenic protein-2.
Hou
L-T
LT
Graduate Institute of Clinical Dentistry, College of Medicine, National Taiwan University, and Department of Periodontology, National Taiwan University Hospital, Taipei, Taiwan. lthou@ha.mc.ntu.edu.tw
Li
T-I
TI
Liu
C-M
CM
Liu
B-Y
BY
Liu
C-L
CL
Mi
H-W
HW
D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects*; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D010012:Osteogenesis / Q000187:drug effects*; D010513:Periodontal Ligament / Q000166:cytology* / Q000201:enzymology; D011994:Recombinant Proteins / Q000494:pharmacology*; D044967:Serum / Q000737:chemistry; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D003470:Culture Media; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D000469:Alkaline Phosphatase