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Open Access Research article

Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

Arthur A DeCarlo1*, Maria Belousova1, April L Ellis1, Donald Petersen1, Hernan Grenett1, Patrick Hardigan2, Robert O’Grady3, Megan Lord3 and John M Whitelock3

Author affiliations

1 Agenta Biotechnologies, Inc, 1500 1st Ave. N., Unit 31, Birmingham, AL, 35203, USA

2 Graduate School of Biomedical Engineering, University of New South Wales, Sydney, Australia

3 Statistical Consulting Center, Nova Southeastern University, 3200 South University Dr, Ft. Lauderdale, FL, 33328, USA

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Citation and License

BMC Biotechnology 2012, 12:60  doi:10.1186/1472-6750-12-60

Published: 11 September 2012

Abstract

Background

Many growth factors, such as bone morphogenetic protein (BMP)-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS) glycosaminoglycans (GAGs), which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS), regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo.

Results

Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1) expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse™).

Conclusions

A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid significantly improved the dose-effectiveness of BMP-2 osteogenic activity for in vivo de novo bone generation when delivered together on a scaffold as a single-phase. The use of HS/CS PGs may be useful to augment GF therapeutics, and a plasmid-based approach has been shown here to be highly effective.

Keywords:
Osteogenesis; BMP-2; Heparan sulfate; Chondroitin sulfate; Proteoglycan; TCP; Bone graft; Implant; Osteoblast; Perlecan