Quantification of rhBMP2 in bioactive bone materials

Bone morphogenetic protein(BMP),belongs to transforming growth factor-b(TGF-b)superfamily except BMP-1.Implanting BMP into muscular tissues induces ectopic bone formation at the site of implantation,which provides opportunity for the treatment of bone defects.Recombinant human BMP-2(rhBMP-2)has been used clinically,but the lack of standard methods for quantifying rhBMP-2 biological activity greatly hindered the progress of commercialization.In this article,we describe an in vitro rhBMP-2 quantification method,as well as the data analyzation pipeline through logistic regression in RStudio.Previous studies indicated that alkaline phosphatase(ALP)activity of C2C12 cells was significantly increased when exposed to rhBMP-2,and showed dose-dependent effects in a certain concentration range of rhBMP-2.Thus,we chose to quantify ALP activity as an indicator of rhBMP-2 bioactivity in vitro.A sigmoid relationship between the ALP activity and concentration of rhBMP-2 was discovered.However,there are tons of regression models for such a non-linear relationship.It has always been a major concern for researchers to choose a proper model that not only fit data accurately,but also have parameters representing practical meanings.Therefore,to fit our rhBMP-2 quantification data,we applied two logistic regression models,three-parameter log-logistic model and four-parameter log-logistic model.The four-parameter log-logistic model(adj-R2>0.98)fits better than three-parameter log-logistic model(adj-R2>0.75)for the sigmoid curves.Overall,our results indicate rhBMP-2 quantification in vitro can be accomplished by detecting ALP activity and fitting four-parameter log-logistic model.Furthermore,we also provide a highly adaptable R script for any additional logistic models.