Research Article: The addition of a polyglutamate domain to the angiogenic QK peptide improves peptide coupling to bone graft materials leading to enhanced endothelial cell activation

Date Published: March 11, 2019

Publisher: Public Library of Science

Author(s): Nicholas W. Pensa, Andrew S. Curry, Michael S. Reddy, Susan L. Bellis, Christophe Egles.


Vascularization of bone grafts is vital for graft integration and bone repair, however non-autologous graft sources have limited potential to induce angiogenesis. Accordingly, intensive research has focused on functionalizing non-autologous materials with angiogenic factors. In the current study we evaluated a method for coupling an angiogenic peptide to the surface of two clinically-relevant graft materials, anorganic bovine bone (ABB) and synthetic hydroxyapatite (HA). Specifically, the VEGF-derived “QK” peptide was synthesized with a heptaglutamate (E7) domain, a motif that has strong affinity for calcium phosphate graft materials. Compared with unmodified QK, a 4–6 fold enrichment was observed in the binding of E7-modified QK (E7-QK) to ABB and HA. The E7-QK peptide was then assessed for its capacity to stimulate angiogenic cell behaviors. Human umbilical vein endothelial cells (HUVECs) were treated with solutions of either QK or E7-QK, and it was found that QK and E7-QK elicited equivalent levels of cell migration, tubule formation and activation of the Akt and ERK signaling pathways. These data confirmed that the inherent bioactivity of the QK sequence was not diminished by the addition of the E7 domain. We further verified that the activity of E7-QK was retained following peptide binding to the graft surface. HA disks were coated with QK or E7-QK, and then HUVECs were seeded onto the disks. Consistent with the increased amount of E7-QK bound to HA, relative to QK, markedly greater activation of Akt and ERK 1/2 was observed in cells exposed to the E7-QK-coated disks. Taken together, these results suggest that the E7 domain can be leveraged to concentrate angiogenic peptides on graft materials, facilitating delivery of higher peptide concentrations within the graft site. The ability to endow diverse graft materials with angiogenic potential holds promise for augmenting the regenerative capacity of non-autologous bone grafts.

Partial Text

More than 2 million bone grafting procedures are performed each year world-wide [1]. Autologous bone is the ideal graft material for these procedures as it retains the osteoinductive growth factors and cells important for effective graft incorporation. However, autologous bone grafts have a number of disadvantages including the risk of secondary surgery site morbidity, as well as the finite amount of donor bone available [2, 3]. To address these issues, non-autogenous graft materials including allograft, xenograft, and synthetic substrates are commonly used as alternatives [4]. These materials are abundant, however, they often lack the critical osteoinductive factors necessary for stimulating graft integration into the surrounding tissue [5]. Without these factors, the potential for complete bone repair is diminished.

The functionalization of non-autologous bone graft materials with bioactive factors constitutes a highly active area of research. Both osteoinductive and angiogenic factors have been investigated for their potential to improve graft performance, however better methods are needed for coupling these factors to the graft surface [43–46]. In the current study we evaluated a method for increasing the binding of an angiogenic peptide, QK, to the surface of calcium phosphate materials. By adding an E7 domain to the QK peptide, we achieved a 4-6-fold enrichment in the amount of peptide loaded onto two graft materials used in the clinic, ABB and synthetic HA. Similar results were reported by Lee et al., who showed that QK binding to HA biomaterials could be enhanced by adding an HA-binding sequence derived from osteocalcin [43]. In tandem with HA binding domains, the QK peptide has been engineered with sequences that have affinity for other bone matrix molecules such as collagen I [47]. As an alternative to peptides with matrix binding domains, soluble QK peptides have been encapsulated within hydrogels [39, 48, 49]. Upon implantation, QK either diffuses from the hydrogel, or is released as the hydrogel degrades. While peptide-containing hydrogels have many worthwhile features, bone grafting procedures often require the use of mineralized materials, which have greater mechanical strength, and offer architectural and biochemical properties reflective of native bone [50, 51].




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