elastin and isoleucyl-lysyl-valyl-alanyl-valine

Spatiotemporal control of vascularization and innervation is a desired hallmark in advanced tissue regeneration. For this purpose, we design a 3D model scaffold, based on elastin-like recombinamer (ELR) hydrogels. This contains two interior and well-defined areas, small cylinders, with differentiated bioactivities with respect to the bulk. Both are constructed on a protease sensitive ELR with a fast-proteolyzed domain, but one bears a VEGF-mimetic peptide (QK) and the other a laminin-derived pentapeptide (IKVAV), to promote angiogenesis and neurogenesis, respectively. The outer bulk is based on a slow proteolytic sequence and RGD cell adhesion domains. In vitro studies show the effect of QK and IKVAV peptides on the promotion of endothelial cell and axon spreading, respectively. The subcutaneous implantation of the final 3D scaffold demonstrates the ability to spatiotemporally control angiogenesis and neurogenesis in vivo. Specifically, the inner small cylinder containing the QK peptide promotes fast endothelialization, whereas the one with IKVAV peptide promotes fast neurogenesis. Both, vascularization and innervation take place in advance of the bulk scaffold infiltration. This scaffold shows that it is possible to induce vascularization and innervation in predetermined areas of the scaffold well ahead to the bulk infiltration. That significantly increases the efficiency of the regenerative activity.

Topics: Elastin; Hydrogels; Laminin; Neurogenesis; Peptide Hydrolases; Peptides; Vascular Endothelial Growth Factor A

Despite significant progress in the field of biomaterials for bone repair, the lack of attention to the vascular and nervous networks within bone implants could be one of the main reasons for the delayed or impaired recovery of bone defects. The design of innovative biomaterials should improve the host capacity of healing to restore a functional tissue, taking into account that the nerve systems closely interact with blood vessels in the bone tissue. The aim of this work is to develop a cell-free and growth factor-free hydrogel capable to promote angiogenesis and innervation. To this end, we have used elastin-like polypeptides (ELPs), poly(ethylene glycol) (PEG) and increasing concentrations of the adhesion peptide IKVAV (25% (w/w) representing 1.7 mM and 50% (w/w) representing 4.1 mM) to formulate and produce hydrogels. When characterized in vitro, hydrogels have fine-tunable rheological properties, microporous structure and are biocompatible. At the biological level, 50% IKVAV composition up-regulated Runx2, Osx, Spp1, Vegfa and Bmp2 in mesenchymal stromal cells and Tek in endothelial cells, and sustained the formation of long neurites in sensory neurons. When implanted subcutaneously in mice, hydrogels induced no signals of major inflammation and the 50% IKVAV composition induced higher vessel density and formation of nervous terminations in the peripheral tissue. This novel composite has important features for tissue engineering, showing higher osteogenic, angiogenic and innervation potential in vitro, being not inflammatory in vivo, and inducing angiogenesis and innervation subcutaneously. STATEMENT OF SIGNIFICANCE: One of the main limitations in the field of tissue engineering remains the sufficient vascularization and innervation during tissue repair. In this scope, the development of advanced biomaterials that can support these processes is of crucial importance. Here, we formulated different compositions of Elastin-like polypeptide-based hydrogels bearing the IKVAV adhesion sequence. These compositions showed controlled mechanical properties, and were degradable in vitro. Additionally, we could identify in vitro a composition capable to promote neurite formation and to modulate endothelial and mesenchymal stromal cells gene expression, in view of angiogenesis and osteogenesis, respectively. When tested in vivo, it showed no signs of major inflammation and induced the formation of a highly vascularized and innervated neotissue. In this sense, our

Topics: Angiogenesis Inducing Agents; Animals; Biocompatible Materials; Bone Morphogenetic Protein 2; Cell Proliferation; Core Binding Factor Alpha 1 Subunit; Elastin; Endothelial Cells; Gene Expression Regulation; Humans; Hydrogels; Laminin; Mesenchymal Stem Cells; Mice; Neurons; Osteogenesis; Osteopontin; Peptide Fragments; Peptides; Polyethylene Glycols; Porosity; Prosthesis Implantation; Rats, Wistar; Rheology; Sp7 Transcription Factor; Surface Properties; Tissue Engineering; Tissue Scaffolds; Vascular Endothelial Growth Factor A

Elastin-like polypeptides (ELPs) are biocompatible-engineered polypeptides, with promising interest in tissue engineering due to their intrinsic biological and physical properties, and their ease of production. The IKVAV (Ile-Lys-Val-Ala-Val) laminin-1 sequence has been shown to sustain neuron attachment and growth. In this study, the IKVAV adhesion sequence, or a scrambled VKAIV sequence, were incorporated by genetic engineering in the structure of an ELP, expressed in Escherichia coli and purified. The transition temperatures of the ELP-IKVAV and ELP-VKAIV were determined to be 23 °C. Although the phase transition was fully reversible for ELP-VKAIV, we observed an irreversible aggregation for ELP-IKVAV. The corresponding aggregates shared some characteristics with amyloid-like polypeptides. The two ELPs were then reacted with functionalized polyethylene glycol (PEG) to form hydrogels. These hydrogels were characterized for rheological properties, tested with cultures of rat primary sensory neurons, and implanted subcutaneously in mice for 4 weeks. Sensory neurons cultured on high IKVAV concentration hydrogels (20%) formed longer neurite than those of neurons grown on hydrogels containing the scrambled IKVAV sequence. Finally, in vivo evaluation showed the absence of detectable inflammation. In conclusion, this functionalized ELP-IKVAV biomaterial shows interesting properties for tissue engineering requiring neurotization.

Topics: Amino Acid Sequence; Animals; Elastin; Hydrogels; Laminin; Peptide Fragments; Peptides; Rats; Rheology; Sensory Receptor Cells; Tissue Engineering