Facile and selective covalent grafting of an RGD-peptide to electrospun scaffolds improves HUVEC adhesion.

The development of a biomimetic surface able to promote endothelialization is fundamental in the search for blood vessel substitutes that prevent the formation of thrombi or hyperplasia. This study aims at investigating the effect of functionalization of poly-ε-caprolactone or poly(L-lactic acid-co-ɛ-caprolactone) electrospun scaffolds with a photoreactive adhesive peptide. The designed peptide sequence contains four Gly-Arg-Gly-Asp-Ser-Pro motifs per chain and a p-azido-Phe residue at each terminus. Different peptide densities on the scaffold surface were obtained by simply modifying the peptide concentration used in pretreatment of the scaffold before UV irradiation. Scaffolds of poly-ε-caprolactone embedded with adhesive peptides were produced to assess the importance of peptide covalent grafting. Our results show that the scaffolds functionalized with photoreactive peptides enhance adhesion at 24 h with a dose-dependent effect and control the proliferation of human umbilical vein endothelial cells, whereas the inclusion of adhesive peptide in the electrospun matrices by embedding does not give satisfactory results.

Osteogenic properties of a short BMP-2 chimera peptide.

Bone morphogenetic proteins (BMPs) play a key role in bone and cartilage formation. For these properties, BMPs are employed in the field of tissue engineering to induce bone regeneration in damaged tissues. To overcome drawbacks due to the use of entire proteins, synthetic peptides derived from their parent BMPs have come out as promising molecules for biomaterial design. On the structural ground of the experimental BMP-2 receptor complexes reported in the literature, we designed three peptides, reproducing the BMP-2 region responsible for the binding to the type II receptor, ActRIIB. These peptides were characterized by NMR, and the structural features of the peptide-receptor binding interface were highlighted by docking experiments. Peptide-receptor binding affinities were analyzed by means of ELISA and surface plasmon resonance techniques. Furthermore, cellular assays were performed to assess their osteoinductive properties. A chimera peptide, obtained by combining the sequence portions 73-92 and 30-34 of BMP-2, shows the best affinity for ActRIIB in the series and represents a good starting point for the design of new compounds able to reproduce osteogenic properties of the parent BMP-2.

Synthesis and chromatography-free purification of PNA-PEO conjugates for the functionalisation of gold sensors.

Peptide Nucleic Acids (PNAs) linked to high molecular weight (MW) poly(ethylene oxide) (PEO) derivatives could be useful conjugates for the direct functionalisation of gold surfaces dedicated to Surface Plasmon Resonance (SPR)-based DNA sensing. However their use is hampered by the difficulty to obtain them through a convenient and economical route. In this work we compared three synthetic strategies to obtain PNA-high MW PEO conjugates composed of (a) a 15-mer PNA sequence as the probe complementary to genomic DNA of ]Mycobacterium tuberculosis, (b) a PEO moiety (2 or 5 KDa MW) and (c) a terminal trityl-protected thiol necessary (after acidic deprotection) for grafting to gold surfaces. The 15-mer PNA was obtained by solid-phase synthesis. Its amino terminal group was later condensed to bi-functional PEO derivatives (2 and 5 KDa MW) carrying a Trt-cysteine at one end and a carboxyl group at the other end. The reaction was carried out either in solution, using HATU or PyOxim as coupling agents, or through the solid-phase approach, with 49.6%, 100% and 5.2% yield, respectively. A differential solvent extraction strategy for product purification without the need for chromatography is described. The ability of the 5 KDa PEO conjugate to function as a probe for complementary DNA detection was demonstrated using a Grating-Coupling Surface Plasmon Resonance (GC-SPR) system. The optimized PEO conjugation and purification protocols are economical and simple enough to be reproduced also within laboratories that are not highly equipped for chemical synthesis.

Self-assembling peptide-enriched electrospun polycaprolactone scaffolds promote the h-osteoblast adhesion and modulate differentiation-associated gene expression.

Electrospun polycaprolactone (PCL) is able to support the adhesion and growth of h-osteoblasts and to delay their degradation rate to a greater extent with respect to other polyesters. The drawbacks linked to its employment in regenerative medicine arise from its hydrophobic nature and the lack of biochemical signals linked to it. This work reports on the attempt to add five different self-assembling (SA) peptides to PCL solutions before electrospinning. The hybrid scaffolds obtained had regular fibers (SEM analysis) whose diameters were similar to those of the extracellular matrix, more stable hydrophilic (contact angle measurement) surfaces, and an amorphous phase constrained by peptides (DSC analysis). They appeared to have a notable capacity to promote the h-osteoblast adhesion and differentiation process by increasing the gene expression of alkaline phosphatase, bone sialoprotein, and osteopontin. Adding an Arg-Gly-Asp (RGD) motif to a self-assembling sequence was found to enhance cell adhesion, while the same motif condensed with a scrambled sequence did not, indicating that there is a cooperative effect between RGD and 3D architecture created by the self-assembling peptides. The study demonstrates that self-assembling peptide scaffolds are still able to promote beneficial effects on h-osteoblasts even after they have been included in electrospun polycaprolactone. The possibility of linking biochemical messages to self-assembling peptides could lead the way to a 3D decoration of fibrous scaffolds.

Cardiomyocytes in vitro adhesion is actively influenced by biomimetic synthetic peptides for cardiac tissue engineering.

Scaffolds for tissue engineering must be designed to direct desired events such as cell attachment, growth, and differentiation. The incorporation of extracellular matrix-derived peptides into biomaterials has been proposed to mimic biochemical signals. In this study, three synthetic fragments of fibronectin, vitronectin, and stromal-derived factor-1 were investigated for the first time as potential adhesive sequences for cardiomyocytes (CMs) compared to smooth muscle cells. CMs are responsive to all peptides to differing degrees, demonstrating the existence of diverse adhesion mechanisms. The pretreatment of nontissue culture well surfaces with the (Arginine-Glycine-Aspartic Acid) RGD sequence anticipated the appearance of CMs' contractility compared to the control (fibronectin-coated well) and doubled the length of cell viability. Future prospects are the inclusion of these sequences into biomaterial formulation with the improvement in cell adhesion that could play an important role in cell retention during dynamic cell seeding.

Chemoselective surface immobilization of proteins through a cleavable peptide.

Surface immobilization of biomolecules is a fundamental step in several experimental techniques such as surface plasmon resonance analysis and microarrays. Oxime ligation allows reaching chemoselective protein immobilization with the retention of native-like conformation by proteins. Beside the need for chemoselective ligation of molecules to surface/particle, equally important is the controlled release of the immobilized molecules, even after a specific binding event. For this purpose, we have designed and assessed in an SPR experiment a peptide linker able to (i) anchor a given protein (enzymes, receptors, or antibodies) to a surface in a precise orientation and (ii) release the immobilized protein after selective enzymatic cleavage. These results open up the possibility to anchor to a surface a protein probe leaving bioactive sites free for interaction with substrates, ligands, antigens, or drugs and successively remove the probe-ligand complex by enzymatic cleavage. This peptide linker can be considered both an improvement of SPR analysis for macromolecular interaction and a novel strategy for drug delivery and biomaterial developments.

Electrospun scaffolds of self-assembling peptides with poly(ethylene oxide) for bone tissue engineering.

Structural, mechanical and biochemical properties have to be considered when searching for suitable extracellular matrix substitutes. Fibrous structures of synthetic or natural polymers have received increasing interest as three-dimensional scaffolds for tissue engineering applications as they can be easily produced by electrospinning with different topographical features by changing the process parameters. On the other hand, the nanobiotechnology approach suggests mimicking molecular architectures in nature through self-assembly. In particular, self-assembling peptide-based biomaterials have been successfully used as scaffolds for cell growth. In order to amalgamate these two strategies nanofibrous electrospun scaffolds of hybrid polymer were designed and obtained by mixing poly(ethylene oxide) and self-assembling peptides in aqueous solution. The results of in vitro osteoblast adhesion and proliferation assays on the electrospun scaffolds obtained using different self-assembling peptide sequences are discussed.