Generation of optimized preparations of bone morphogenetic proteins for bone regeneration.

Kh V Malysheva,I M Spasyuk,R S Stoika,O G Korchynsky,O K Pavlenko

doi:10.15407/ubj88.06.087

Abstract

Correction of inherited skeletal abnormalities, traumas affecting wide bone areas and non-healing fractures require efficient bone formation and regeneration. Bone morphogenetic proteins (BMPs) are signaling molecules that play a crucial role in bone and cartilage formation and regeneration. Osteoinductive properties of existing hydroxyapatite-based osteoplastic materials are frequently insufficient for efficient bone regeneration, thus raising a requirement for novel matrices involving BMPs for highly efficient local induction of bone formation at the area of the bone defect. The aim of this study was conducting in vitro optimization of osteoinductive properties of recombinant BMPs preparations to be used in bone regenerative practice. Recombinant BMPs were produced in human embryonic kidney 293 cells upon their transfection or co-transfection with plasmids expressing BMP2 and BMP7 at different ratios. A quality of BMP preps was validated based on their ability to induce in vitro osteoblast differentiation of C2C12 cells. Alkaline phosphatase that is widely used as a marker of osteoblast differentiation was measured spectrophotometrically. We found that the most effective inducer of osteoblast differentiation was recombinant BMP preparation produced upon cotransfection of 85% of BMP2 and 15% of BMP7 plasmids, that is most likely due to generation of conditions most favorable for formation of BMP2/7 heterodimers. Frozen BMP2/7 preparations stored for 3 h in experimental setup and for several weeks in routine work do not lose their osteoinductive properties compared with freshly prepared BMP2/7 preparations and can be successfully used for generation of highly efficient bone regenerative matrices.

Highlights

Bone morphogenetic proteins (BMPs) belong to a group of multifunctional signaling molecules that belong to the transforming growth factor-β (TGF-β) superfamily of proteins
While BMPR-1A, BMPR-1B, and BMPR-2 are specific to BMPs, ActR-1A, ActR-2A, and ActR-2B can function as receptors for activins, which are members of the TGF-β superfami ly
Plasmids expressing BMP2 and BMP7 full-length cDNA were purchased from Open Biosystems/GE Healthcare (Lafayette, CO, USA). pShuttle-CMV vector was kindly provided by Dr Bert Vogelstein

Summary

Introduction

In the canonical signaling pathway, they initiate the signal transduction cascade by binding to cell surface receptors and forming a heterotetrameric complex comprised of two dimers of type I and type II serine/ threonine kinase receptors [7]. Both receptor types have a short extracellular domain, a single transmembrane domain, and an intracellular domain with serine/threonine kinase activity. Upon formation of a heterotetrameric complex, the constitutively active type II receptor trans-phosphorylates the type I receptor at a glycine–serine rich motif known as the GS domain This activates the type I receptor and allows phosphorylation of the immediately downstream substrate proteins known as the receptor-regulated Smads (R-Smads) at a C-terminal SSXS motif. Inhibitory Smads (I-Smads), Smad and Smad (Smad6/7), are involved in feedback inhibition of the signaling pathway [4, 10, 11]

Objectives

Methods

Results