Gene Delivery of Heparan Sulfate Proteoglycan Core Enhances Bone Regeneration

Heparan sulfate, a glycosaminoglycan related to heparin, is found O-linked to a subset of proteoglycan core proteins, such as perlecan. Heparan sulfate of perlecan enables the binding of fibroblast growth factors (FGFs), presentation of FGF to receptor pairs, and activation of downstream signaling cascades. Promotion of angiogenesis, cell differentiation, and cell growth are attributed to heparan sulfate proteoglycans, perlecan in particular. Objectives: 1) to create a eukaryotic expression construct to generate the heparan sulfate domain of the proteoglycan perlecan, and 2) to measure the effects of co-delivering this perlecan transgene expression construct with hydroxyl apatite bone graft material in an animal model for bone regeneration. Methods: RT-PCR of endothelial cell total RNA generated a 600 bp transgene of the perlecan N-terminal domain 1 (D1) region which was delivered in vitro and in vivo via a replication-defective adenovirus. Antibodies specific for perlecan and glycosaminoglycans were used to evaluate recombinant expression, while bone healing was measured as density of backlit calvarial specimens. Results: Recombinant perlecan D1 was detected only in the conditioned medium of cell culture and demonstrated 1) heparan sulfate glycosylation, 2) an apparent kDa of 32 –45 in SDS-PAGE, and 3) FGF binding in vitro. The adenovirus adsorbed tightly to hydroxyl apatite (HA) crystals in neutral buffer, and co-delivery of HA with approximately 105 adenoviral units to a 3 mm round calvarial defect in the mouse resulted in significantly denser bone after 18 days relative to paired control sites (p = .05, paired t-test) with histological confirmation. Conclusion: a functional, glycosylated recombinant perlecan D1 can be independently expressed as a soluble proteoglycan, and transient upregulation of perlecan D1 within an osseous defect may be a safe and useful adjunct to craniofacial bone regenerative procedures. This work was supported by a grant (R44 DE016771) from the NIDCR.