A reporter‐cell assay for the detection of BMP‐2 immobilized on porous and nonporous materials

Abstract

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.

Heat‐treated BMP‐2 depots release BMP‐2 in its bioactive form

“…BMP-2 depots on glass: Cover slips were cleaned in 5 % HNO 3 (80 C), and further processed as described [4]. Up to 3 droplets (1ll) of BMP-2 stock solution were air-dried onto the glass surface, exposed to 100 C for 30 min, and stored for up to 3 d until use.…”

“…Histochemical detection of ALP: To visualize cell bound ALP, MC3T3-E1 cells were fixed with 4 % paraformaldehyde in 0.1 mol/l sodium phosphate buffer (PBS, pH 7.4), washed 3 times with PBS, and immersed in 100 mM Tris-buffered staining solution (pH 10.4) containing Nitro Blue Tetrazoleum and 5-bromo-4-chloro-3-indolyl phosphate as a chromogene [4]. Colour development was stopped by washing 3 times with PBS after 30 minutes.…”

“…Cultured directly on BMP-2 depots, these cells up-regulate fibronectin and actin as well as bone differentiation markers such as osteocalcin, collagene I, and alkaline phosphatase (ALP). All these changes in protein expression were largely confined to the BMP-2 coated area [4]. This and the fact that recombinant BMP-2 poorly dissolves in aquaeous solution [5] suggested so far that BMP-2 receptors were stimulated by immobilized BMP-2.…”

“…Since BMP-2 has various inductive properties also for other tissues [2], and because relatively high amounts of the protein are needed for a successful formation of bone in man [1,3], a spatially confined application of BMP-2 appears desirable for a directed and controlled induction of bone. Recently, we have shown that BMP-2, which was air-dried onto various metal implant materials such as 316L stainless steel or titanium, showed an augmented bioactivity if additionally heated to 100 o C for 30 min [4]. Locally immobilized BMP-2 treated this way will be termed "BMP-2 depot" hereafter.…”

How to use BMP-2 for clinical applications? A review on pros and cons of existing delivery strategies

“…For this reason biotin and streptavidin are a very convenient choice for protein immobilization. In context of BMP-2 immobilization, biotin-streptavidin interaction has been used to study the biological activity of bound BMP-2 [105], as well as for quantifying small amounts of immobilized BMP-2 on various materials [106]. Recently, a continuous surface BMP-2 gradient was constructed using biotin-streptavidin interaction, to enable cell screening studies [107].…”