The effect of non-collagenous proteins on BMP retention and activity

Background: Partially purified BMP (BMP/NCP) is more stable and has prolonged effects in cell culture compared to recombinantly produced BMP (rBMP). We hypothesized that the non-BMP proteins in BMP/NCP bind and slowly release the BMP.

Objectives: To determine the ability of substrata coated with various proteins to retain BMP and their effect on BMP activity.

Methods: 96 well plates were coated with BSA, collagen, noggin, fetuin, or left uncoated. rhBMP-2 was added to the wells and air dried. The plates were incubated with PBS+0.1% BSA and the BMP released was measured by ELISA.

BMP activity was determined as follows: 24 well plates were coated with the test proteins. Cell culture medium, with and without rhBMP-2 was added. Some wells were then rinsed with fresh medium without BMP to remove any unbound BMP. C2C12 cells were seeded onto the plates, cultured for 2 or 5 days, and assayed for alkaline phosphatase (ALP) activity. Results were analysed by ANOVA.

Results: The amount of rhBMP-2 released from noggin coated wells was significantly lower that that released from any of the other coatings (P<0.001). Addition of BMP resulted in an increase in ALP activity in cultures on all substrata (P<0.001). Cells cultured on collagen or noggin coated plates had the greatest increase in ALP with rhBMP-2 treatment. In cultures where BMP was rinsed away prior to cell seeding, elevated ALP levels were only seen in the noggin coated wells (P<0.001). However when Noggin was added to BMP in solution, ALP activity was inhibited.

Conclusions: Our results demonstrate that noggin binds BMP preventing or delaying its release into buffer solution. In addition, while noggin in solution inhibits BMP activity, substrata coated with noggin bind BMP but retained its activity.