Bone morphogenetic protein-2 (BMP-2)

one of the important osteoinductive growth factors

is an effective and crucial cytokine for clinic bone regeneration as well as bioactivity improvement of bone substitute. However

extraction of BMP-2 from animals can hardly meet the clinic requirements due to its very low content. This work studied the preparation of BMP-2 to meet the clinical needs

and evaluated its biological activity. Firstly

DNA sequence of optimized hBMP-2 gene was obtained to prepare rhBMP-2 strains of E. coli using codon optimization method with further replacement of the partial nucleotide coding. Then

through fermentation and process optimization the BMP inclusion body was acquired. After separation

purification and renaturation

rhBMP-2 of high purity was prepared. In vitro cell experiments

the activity of alkaline phosphatase (ALP) in C2C12 cells was measured to characterize the osteogenic activity of haploid and diploid BMP-2. It was observed that the osteogenic activity of diploid rhBMP-2 was significantly higher than that of haploid rhBMP-2

and the activity of alkaline phosphatase increased with the increase of the BMP-2 concentration. In vivo ectopic bone experiment

after rhBMP-2 was implanted into the muscle of mice for 3 weeks

the ectopic bone granules were bright and fresh

and the bone structure was complete. HE and Masson's trichrome stainings showed good ectopic bone formation. It can be concluded that the rhBMP-2 prepared using this method can well induce osteogenic differentiation

and can be used for bone tissue repair to meet the clinical needs.