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.

Site-specific delivery of acidic fibroblast growth factor stimulates angiogenic and osteogenic responses in vivo.

A major clinical problem in orthopedics is the healing of nonunion fractures. Limitations of this bone repair process include insufficient angiogenesis and mineralization. Integrating appropriate biomaterials with site-specific neovascularization and osteogenesis at the wound site has been the focus of several clinically relevant therapeutic strategies. As an extracellular protein, acidic fibroblast growth factor (FGF-1) induces, coordinates, and sustains site-specific molecular responses associated with angiogenesis and osteogenesis. To establish the ability of this growth factor to coordinate bone regenerative process in vivo, site-specific delivery of FGF-1, entrapped in a fibrin/hydroxyapatite composite, was evaluated. Kinetic analysis in vivo revealed the biocomposite was capable of delivering biologically active FGF-1. Release kinetics revealed an initial delivery of 87.5 ng/h of active FGF-1 in the first 20 h, followed by a reduced delivery of 28 ng/h during the next 20 h. In situ immunohistological analyses demonstrated that FGF-1-containing implants induced increased angiogenesis and infiltration of cells expressing osteogenic related markers (i.e., osteopontin, osteocalcin). Collectively, these efforts support that site-specific delivery of active FGF-1 in a fibrin/hydroxyapatite composite is competent to induce not only angiogenesis but also osteogenic cellular responses.

Acidic fibroblast growth factor signaling inhibits peroxynitrite-induced death of osteoblasts and osteoblast precursors.

After trauma injury to the musculoskeletal system, conditions such as ischemia and inflammation involve excess production of superoxide (O2*), nitric oxide (*NO), and their reaction product, peroxynitrite (ONOO-). Exposure of murine osteoblasts and rat-derived primary osteoblast precursors to ONOO- resulted in a dose- and time-dependent delayed cell death that was more characteristic of apoptosis than necrosis. Exposure of both cell populations to ONOO- immediately enhanced phosphorylation and nitration of tyrosine residues within several polypeptides. Treatment of osteoblasts and osteoblast precursors with exogenous acidic fibroblast growth factor (FGF-1) enhanced cellular growth, increased endogenous levels of tyrosine phosphorylation, and significantly induced expression of both osteopontin and osteocalcin messenger RNA (mRNA) as well as osteopontin protein. Pretreatment of both cell populations with exogenous FGF-1 prevented ONOO(-)-mediated death. Cell signaling induced by FGF-1 pretreatment had no major effect of total levels of tyrosine nitration after ONOO- treatment. Collectively, these in vitro efforts show that FGF-1 signaling renders osteoblasts and osteoblast precursors resistant to the cytotoxic effects of ONOO-. Consequently, results presented here predict the therapeutic use of this growth factor for promoting the progression of bone repair mechanisms after fracture trauma.

Estrogen attenuates integrin-beta(3)-dependent adventitial fibroblast migration after inhibition of osteopontin production in vascular smooth muscle cells.

BACKGROUND: Previous in vitro studies have suggested that estrogen attenuates the vascular injury response by modulating vascular smooth muscle cell (VSMC) expression of soluble factor(s) directing migration of adventitial fibroblasts. Previous in vivo studies have established a role for osteopontin (OPN) and its integrin receptors after vascular injury. In this study, we examined OPN expression in activated VSMCs, its modulation by estrogen, and its effects on adventitial fibroblast migration. In addition, the relative functional roles of beta(1)- and beta(3)-integrin-matrix interactions were examined. METHODS AND RESULTS: Primary cultures of VSMCs and adventitial fibroblasts were derived from female Sprague-Dawley rats. Serum-activated VSMCs expressed high levels of OPN mRNA and secreted protein that was effectively inhibited by estrogen treatment (10(-7) mol/L). Compared with VSMCs, fibroblasts expressed similar levels of integrins alphanu and beta(1) and higher levels of integrin-beta(3). Exogenous OPN (5.0 to 40 microg/mL) directed fibroblast migration in a dose-dependent fashion. Anti-beta(3)-integrin antibody (F11) pretreatment markedly inhibited adventitial fibroblast migration directed by exogenous OPN or VSMC-conditioned medium in a dose-dependent manner. In contrast, anti-beta(1)-integrin antibody (Ha2/5) did not affect fibroblast migration. Similarly, pretreatment with either linear or cyclic RGD peptides (10 to 1000 micromol/L) inhibited fibroblast migration directed by OPN or VSMC-conditioned medium in a dose-dependent manner. CONCLUSIONS: These observations suggest that estrogen indirectly attenuates integrin-beta(3)-dependent adventitial fibroblast migration after inhibition of OPN expression in VSMCs.