In vitro evaluation of recombinant bone morphogenetic protein-2 bioactivity for regenerative medicine

Stephanie L. Fung, Xiaohuan Wu, Julian P. Maceren, Yong Mao, Joachim Kohn

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a commonly used growth factor in bone regeneration due to its high potency and ability to induce osteogenic differentiation of osteoblasts and osteoblast precursors. When designing delivery systems for rhBMP-2, the activity of the loaded and released protein is an important consideration. The variability in the experimental design parameters used to measure rhBMP-2 activity in vitro has precluded comparative analysis. Here, for the first time, we report a direct comparison of the assay parameters used in rhBMP-2 bioactivity assays in the literature and an evaluation of commercially available rhBMP-2 obtained from different vendors. Most published rhBMP-2 assays use W-20-17 (mouse stromal), MC3T3 (preosteoblast), or C2C12 (myoblast) cell lines. We found that each model cell line has an optimal concentration range over which it is most sensitive to rhBMP-2 induction. Therefore, it is difficult to find one single bioassay protocol that could be universally used. In addition, we established a correlation between protein concentration (as measured by enzyme-linked immunosorbent assay) and protein activity (as measured by alkaline phosphatase induction). We found that the expression system used to produce the rhBMP-2 had the greatest effect on its activity and stability in vitro. Establishing a standard method of measuring rhBMP-2 activity in vitro is the first step toward developing an in vitro-in vivo correlation between measured activity and clinical outcomes. This work is a systematic evaluation of the experimental parameters of the most widely used in vitro recombinant human bone morphogenetic protein-2 (rhBMP-2) activity assays. The variations in assays reported in the literature have challenged the reproducibility and translation of work using rhBMP-2 as a bone-inducing growth factor. By elucidating the effect of model cell line on the dose-dependent alkaline phosphatase response to rhBMP-2 induction and by establishing a correlation between protein activity and protein concentration by enzyme-linked immunosorbent assay using commercially available rhBMP-2, this work is a significant step toward developing an in vitro-in vivo correlation between quantified activity and clinical efficacy.

Original languageEnglish (US)
Pages (from-to)553-559
Number of pages7
JournalTissue Engineering - Part C: Methods
Issue number9
StatePublished - Sep 2019

All Science Journal Classification (ASJC) codes

  • Bioengineering
  • Medicine (miscellaneous)
  • Biomedical Engineering


  • BMP-2
  • alkaline phosphatase activity
  • bioactivity
  • bone regeneration


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