Strong and Rapid Induction of Osteoblast Differentiation by Cbfa1/Til-1 Overexpression for Bone Regeneration

Toshimasa Uemura,Hiroko Kojima

doi:10.1074/jbc.m311598200

Abstract

Core binding factor alpha-1 (Cbfa1), known as an essential transcription factor for osteogenic lineage, has two major N-terminal isoforms: Pebp2alphaA and Til-1. To study the roles of these isoforms in bone regeneration, we applied an adenoviral vector carrying their genes to transduce primary osteoprogenitor cells in vitro and in vivo. Overexpression of the two isoforms induced rapid and marked osteoblast differentiation, with Til-1 being more effective in vitro, by examination of the alkaline phosphatase activity, calcium content, and Alizarin red staining. Til-1 overexpressing cells/porous ceramic composites were transplanted into subcutaneous and bone defect sites in Fischer rats (cultured bone transplantation model) and markedly affected in vivo bone formation and osteoblast markers. The results demonstrated that the reconstitution of bone tissues, such as cortical bone and trabecular bone was accelerated by implantation of Til-1 overexpressing cells/porous ceramic composites. Moreover, the new bone formation by Til-1 overexpression appeared to reflect replacement of new bone within the implant boundaries. To ascertain whether implanted Cbfa1 overexpressing cells could differentiate into osteogenic cells to create bone or whether it stimulated the surrounding recipient tissue to regenerate bone, implanted male donor cells were visualized by fluorescent in situ hybridization analysis. The proportion of implanted cells in the presumptive bone forming region was over 80% and did not change throughout from 3 days to 8 weeks after implantation. These findings suggested that the newly formed bone in the porous area of the scaffold is mostly produced by the implanted donor cells or their derived cells, effectively by Til-1 overexpression.

Highlights

Core binding factor ␣-1 (Cbfa1), known as an essential transcription factor for osteogenic lineage, has two major N-terminal isoforms: Pebp2␣A and Til-1
In Vitro Effect of Cbfa1 Overexpression in Primary Osteoprogenitor Cells—First, we examined the effect that the overexpression of Cbfa1 has on the differentiation of rat bone marrowderived primary osteoprogenitor cells, cultured by the methods of Maniatopoulos et al (34)
One approach to accelerating the osteoblast differentiation process involves the use of gene therapy in which viral or non-viral vectors are utilized to overexpress osteogenic factors in cells for transplantation

Summary

Introduction

Core binding factor ␣-1 (Cbfa1), known as an essential transcription factor for osteogenic lineage, has two major N-terminal isoforms: Pebp2␣A and Til-1. The procedure essentially involves the in vivo transplantation of MSC-derived osteoprogenitor cells and porous ceramic composite, to form bone-like tissue at ectopic (subcutaneous and intramuscular) or orthotopic sites, providing tissue engineering to patients with skeletal defects. Rat bone marrow-derived osteoprogenitor cells and porous ceramic composite were cultured in vitro with osteogenic supplements: dexamethasone, ␤-glycerophosphate, and ascorbic acid This was followed by a subcutaneous transplantation resulting in new bone forming porous internal areas in vivo. Before this system can be applied clinically, several tasks need to be performed, such as elucidation of the mechanism of in vivo bone formation, improvement of ceramic scaffolds, and the advancement of culture techniques of MSC-derived cells. Osteogenic cytokines like bone morphgenetic protein (19 –21) and transforming growth fac-

Methods

Results

Conclusion