The effect of the addition of a polyglutamate motif to RGD on peptide tethering to hydroxyapatite and the promotion of mesenchymal stem cell adhesion.
Mimicking endogenous bone-binding proteins, RGD peptides have been synthesized with polyacidic amino acid domains in order to ionically tether the peptides to bone-like synthetic biomaterials, including hydroxyapatite (HA). However, a direct comparison of unmodified RGD with polyacidic-conjugated RGD has not been performed, and thus a benefit for the acidic domain has not been established. We evaluated the peptide/HA bond of RGD peptides with and without an attached polyglutamate sequence (E(7)), as well as examined mesenchymal stem cell (MSC) adhesion and morphology as they were affected by the conjugated peptide. We found that significantly more E(7)RGD was bound to HA than RGD at all coating concentrations tested, and moreover, more E(7)RGD was retained on the HA surface even after extended washing in serum-free media. Consistent with in vitro results, higher levels of E(7)RGD than RGD remained on HA that had been implanted in vivo for 24 h, indicating that the polyacidic domain improved peptide-binding efficiency. At several peptide concentrations, E(7)RGD increased cell adhesion compared to RGD surfaces, establishing a biological benefit for the E(7) modification. In addition, HA pre-coated sequentially with low-density E(7)RGD (1-10 microg/ml) and serum (FBS) stimulated cell adhesion and spreading, compared to either coating alone, suggesting that an ionic linkage allows for the potential adsorption of serum proteins to unoccupied sites, which may be important for bone formation in vivo. Collectively, these results suggest that tethering peptides to HA via a polyglutamate domain is an effective method for improving the peptide/HA bond, as well as for enhancing MSC adhesion.
Sawyer
A A
AA
Department of Biomedical Engineering, University of Alabama at Birmingham, 35294, USA.
Weeks
D M
DM
Kelpke
S S
SS
McCracken
M S
MS
Bellis
S L
SL
D000293:Adolescent; D000327:Adsorption; D000328:Adult; D018786:Bone Substitutes / Q000737:chemistry / Q000494:pharmacology*; D002448:Cell Adhesion / Q000187:drug effects*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D020099:Coated Materials, Biocompatible / Q000737:chemistry / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D017886:Durapatite / Q000737:chemistry* / Q000494:pharmacology; D005260:Female; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology*; D010012:Osteogenesis / Q000187:drug effects* / Q000502:physiology; D011099:Polyglutamic Acid / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding
D018786:Bone Substitutes; D020099:Coated Materials, Biocompatible; D009842:Oligopeptides; D011099:Polyglutamic Acid; C047981:arginyl-glycyl-aspartic acid; D017886:Durapatite