[Experiment on induction of fibroblasts on 3-D cell-foam structures to express osteoblastic phenotype and its mechanism].
OBJECTIVE
To study the feasibility of osteogenic phenotype expression by human skin fibroblasts induced in polyglycolic acid (PGA) foams and the effect of tumor necrosis factor-alpha (TNF-alpha) on the expression of bone morphogenetic protein (BMP) receptors.


METHODS
The fibroblasts were isolated, purified from human skin. (1) Fibroblasts were seeded onto PGA foams. The cell-PGA complexes were cultured in RCCS for 6 weeks, in the media of TNF-alpha (50 U/ml) and BMP-2 (0.1 microg/ml). 1 d, 3 and 6 weeks later, cells and extracellular matrix were investigated by electron microscopic and histochemistry observation respectively. Secretion of osteogenic markers were analyzed by biochemical methods. (2) Fibroblasts were seeded on the glass fragments or culture flasks and treated with TNF-alpha (50 U/ml) in different usage (one-time, all-time). The RT-PCR method and the immunohistochemistry fluorescence staining were used to examine the influence of TNF-alpha on the mRNA expression and the protein expression of the type I BMP receptors at 2, 4, 6, 8 d after treatment.


RESULTS
Fibroblasts seeded on the PGA foams formed 3-dimensional matrix 3 weeks after seeding, which was demonstrated as osteo-tissue by tetracycline labeling and ARS staining. Cells secreted much more bone-specific alkaline phosphatase (B-AKP) and osteocalcin (OCN) into supernatant than the cells that were cultured in the media without TNF-a and BMP2. Eight days after all-time usage, the TNF-alpha (50 U/ml) increased the expression of the mRNA and protein of the type IB BMP receptor.


CONCLUSIONS
Fibroblasts on 3-D cell-foam structures can express osteoblastic phenotype under certain inducing conditions. The numerous fibroblasts in body would be a promising resource for cell seeds candidate of tissue- engineered bone. TNF-alpha provides the essential condition for BMP2's target effect on fibroblasts, and combined use of TNF-alpha and BMP2 is one of the regulating factors.
He
Chuan
C
Shanghai Institute of Traumatology and Orthopaedics, Shanghai 200025, China.
Deng
Lian-fu
LF
Yang
Qing-ming
QM
Shen
Wei
W
Feng
Wei
W
Zhang
Yue
Y
Zhu
Ya-ping
YP
D055396:Bone Morphogenetic Protein 2; D052005:Bone Morphogenetic Protein Receptors, Type I / Q000096:biosynthesis / Q000235:genetics*; D052006:Bone Morphogenetic Protein Receptors, Type II / Q000096:biosynthesis / Q000235:genetics*; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D004357:Drug Synergism; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D010641:Phenotype; D011100:Polyglycolic Acid; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; C439524:TNF protein, human; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D011100:Polyglycolic Acid; D052005:Bone Morphogenetic Protein Receptors, Type I; D052006:Bone Morphogenetic Protein Receptors, Type II