A reporter-cell assay for the detection of BMP-2 immobilized on porous and nonporous materials.
Human recombinant bone morphogenetic protein-2 (rhBMP-2) immobilized on the surface of metal implants can facilitate osseointegration. Here, we describe a cell reporter assay useful for quantifying small amounts of immobilized rhBMP-2 on various materials. The peptide was dotted and heat-fixed on titanium, 316L stainless steel, nitrocellulose, or glass, and its distribution was monitored by in situ biotinylation followed by detection with the avidin-biotin method. Bioactivity of rhBMP-2 was demonstrated by means of a confluent layer of osteoblastic MC3T3-E1 cells that evenly covered rhBMP-2-free and rhBMP-2-loaded surface areas, as shown with epifluorescence microscopy of calcein acetoxymethyl (AM)-loaded cells. Expression of osteocalcin, fibronectin, actin, and vimentin increased where cells were located on rhBMP-2 dotted areas, but the signal:noise ratio was too low to bioassay the peptide. However, local pronounced expression of alkaline phosphatase was used to quantify BMP-2 in the range of 5-80 ng/dot by means of a cytochemical color reaction for alkaline phosphatase and image analysis of resulting dots. The lower detection limit was in the order nitrocellulose > glass > titanium > 316L steel. We conclude that the cell reporter assay is useful to assess biological activity of rhBMP-2 even after immobilization on three-dimensional implant materials.
Institut für Physiologie, Universität Essen, Hufelandstr. 55, D-45122 Essen, Germany. martin.wiemann@uni-essen.de
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M
Wiemann
H P
HP
Jennissen
H
H
Rumpf
L
L
Winkler
M
M
Chatzinikolaidou
I
I
Schmitz
D
D
Bingmann
D000469:Alkaline Phosphatase / Q000032:analysis / Q000187:drug effects; D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000032:analysis* / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D020099:Coated Materials, Biocompatible / Q000032:analysis* / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D051379:Mice; D016348:Osseointegration / Q000187:drug effects*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects; D016062:Porosity; D011994:Recombinant Proteins / Q000032:analysis / Q000494:pharmacology; D012680:Sensitivity and Specificity; D016212:Transforming Growth Factor beta
C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D020099:Coated Materials, Biocompatible; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D000469:Alkaline Phosphatase