Hydroxyapatite-Binding Domains Direct Anchoring Of Osteoinductive Peptides To Bone Grafts

Objectives: Autogenous bone is widely used as the gold standard in craniofacial regenerative medicine. However, harvesting bone is associated with increased morbidity and therefore clinicians often turn to allograft materials that may lose their osteoinductivity due to processing.  Re-establishing the osteoinductive potential in allografts would be a major step forward in enhancing clinical outcomes. The aim of this study was to reintroduce osteoinductive factors to three bone grafts.  A collagen-mimetic peptide (DGEA) was used because it is osteoinductive and stimulates differentiation of mesenchymal stem cells into osteoblasts. A highly negative heptaglutamate (E7) domain was utilized to anchor DGEA to hydroxyapatite mineral within the bone graft.

Methods: Equimolar solutions of FITC-tagged E7-DGEA or DGEA were incubated with varying amounts of graft material for 30 minutes.  The depletion of fluorescence from solution (representing peptide) was quantified and subtracted from the values of starting solution to calculate the percent of peptide bound.  Greater binding of E7-DGEA was confirmed by direct visualization of the grafts by fluorescent microscopy. To evaluate peptide release, grafts were loaded with peptides for 30 minutes, washed briefly, and then re-suspended in TBS with agitation for 3 days. The percentage of peptide released was quantified by measuring solution fluorescence.

Results: Direct visualization of grafts, in combination with solution fluorescence depletion assays, confirmed greater binding of E7-DGEA than DGEA to 3 diverse graft materials (Table I).  Furthermore, a greater quantity of E7-DGEA was retained on the grafts after a 3-day incubation with agitation (Table II).

Conclusions: Addition of a heptaglutamate domain to osteoinductive peptides provides an effective mechanism for enhancing peptide loading and retention on multiple types of bone graft materials.  The ability to anchor bioactive molecules to bone grafts for sustained delivery in vivo could potentially increase osteoinductivity of these products and improve regenerative capacity.

	Small particle xenograft		Large particle xenograft		Cortical/cancellous allograft
	DGEA	E7-DGEA	DGEA	E7-DGEA	DGEA	E7-DGEA
12.5mg	56	88	28	94	4	72
25mg	45	85	47	90	2	85
50mg	70	76	65	89	10	88
Table 1: Percent of peptide initially bound to grafts.

 

	Small particle xenograft %Released-3 days		Large particle xenograft %Released-3 days		Cortical/cancellous allograft %Released-3 days
	DGEA	E7-DGEA	DGEA	E7-DGEA	DGEA	E7-DGEA
12.5mg	32	9	47	9	21	23
25mg	25	9	54	7	57	10
50mg	38	11	31	16	63	11
Table 2: Percent of bound peptide released after three days.

This abstract is based on research that was funded entirely or partially by an outside source: NIDCR DE017607

Keywords: Bone, Osteoblasts/osteoclasts and Osteoinductivity

See more of: Bone, Periodontal Ligament, and Stem Cell Biology
See more of: Mineralized Tissue

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