Development of Biomimetic Alginate/Gelatin/Elastin Sponges with Recognition Properties toward Bioactive Peptides for Cardiac Tissue Engineering.

In recent years, there has been an increasing interest toward the covalent binding of bioactive peptides from extracellular matrix proteins on scaffolds as a promising functionalization strategy in the development of biomimetic matrices for tissue engineering. A totally new approach for scaffold functionalization with peptides is based on Molecular Imprinting technology. In this work, imprinted particles with recognition properties toward laminin and fibronectin bioactive moieties were synthetized and used for the functionalization of biomimetic sponges, which were based on a blend of alginate, gelatin, and elastin. Functionalized sponges underwent a complete morphological, physicochemical, mechanical, functional, and biological characterization. Micrographs of functionalized sponges showed a highly porous structure and a quite homogeneous distribution of imprinted particles on their surface. Infrared and thermal analyses pointed out the presence of interactions between blend components. Biodegradation and mechanical properties appeared adequate for the aimed application. The results of recognition tests showed that the deposition on sponges did not alter the specific recognition and binding behavior of imprinted particles. In vitro biological characterization with cardiac progenitor cells showed that early cell adherence was promoted. In vivo analysis showed that developed scaffolds improved cardiac progenitor cell adhesion and differentiation toward myocardial phenotypes.

Functionalization of Electrospun Nanofibers and Fiber Alignment Enhance Neural Stem Cell Proliferation and Neuronal Differentiation.

Neural stem cells (NSCs) have the potential to generate the cells of the nervous system and, when cultured on nanofiber scaffolds, constitute a promising approach for neural tissue engineering. In this work, the impact of combining nanofiber alignment with functionalization of the electrospun poly-ε-caprolactone (PCL) nanofibers with biological adhesion motifs on the culture of an NSC line (CGR8-NS) is evaluated. A five-rank scale for fiber density was introduced, and a 4.5 level, corresponding to 70-80% fiber density, was selected for NSC in vitro culture. Aligned nanofibers directed NSC distribution and, especially in the presence of laminin (PCL-LN) and the RGD-containing peptide GRGDSP (PCL-RGD), promoted higher cell elongation, quantified by the eccentricity and axis ratio. In situ differentiation resulted in relatively higher percentage of cells expressing Tuj1 in PCL-LN, as well as significantly longer neurite development (41.1 ± 1.0 µm) than PCL-RGD (32.0 ± 1.0 µm), pristine PCL (25.1 ± 1.2 µm), or PCL-RGD randomly oriented fibers (26.5 ± 1.4 µm), suggesting that the presence of LN enhances neuronal differentiation. This study demonstrates that aligned nanofibers, functionalized with RGD, perform as well as PCL-LN fibers in terms of cell adhesion and proliferation. The presence of the full LN protein improves neuronal differentiation outcomes, which may be important for the use of this system in tissue engineering applications.

Neuronal production from induced pluripotent stem cells in self-assembled collagen-hyaluronic acid-alginate microgel scaffolds with grafted GRGDSP/Ln5-P4.

Self-assembled microgel functionalized with peptides was developed and applied to regenerate neurons from induced pluripotent stem cells (iPSCs). Collagen (COL), hyaluronic acid (HA), and alginate (ALG) were modified with methacrylic anhydride (MA), photocrosslinked for patterned particles, grafted with GRGDSP and Ln5-P4, and self-assembled to integrate the microgel into three-dimensional scaffolds. Physicochemical assessments revealed that the ternary microgel scaffolds had an optimal chemical composition at COLMA:HAMA:ALGMA=1:2:1. In fabricating cell-laden constructs, modified GRGDSP/Ln5-P4 in linear self-assembled scaffolds could significantly improve the entrapment efficiency and viability of iPSCs. In addition, GRGDSP/Ln5-P4 in the microgel constructs triggered the differentiation of iPSCs toward neurons, since the percentage of neurite-like cells could be higher than 98% after induction of nerve growth factor. Self-assembled microgel comprising COLMA, HAMA, ALGMA, and GRGDSP/Ln5-P4 may be promising in producing mature neural lineage from iPSCs, to provide better treatment for damaged nervous tissue.

Sandblasting and fibronectin-derived peptide immobilization on titanium surface increase adhesion and differentiation of osteoblast-like cells (MC3T3-E1).

BACKGROUND/PURPOSE: Various chemical titanium (Ti) surface modifications have been reported for enhancing cellular activities that promote early osseointegration. The purpose of this study was to determine if sandblasted Ti coated with or without fibronectin (FN) or FN-derived peptides stimulated osteoblast-like cell adhesion, spreading, proliferation, and differentiation. MATERIALS AND METHODS: Osteoblast-like cells (MC3T3-E1) were cultured on sandblasted Ti disks immobilized with FN or FN-derived peptides [GRGDSP (Gly-Arg-Gly-Asp-Ser), PHSRN (Pro-His-Ser-Arg-Asn), or GRGDSP/PHSRN]. Surface topography, cell morphology, cell adhesion, cell proliferation, analysis of osteogenesis-related genes and protein expression, alkaline phosphatase, and alizarin red staining of mineralization were evaluated. RESULTS: The sandblasted Ti coated with FN or FN-derived peptides enhanced cell adhesion and cell proliferation. However, the Ti coated with FN or FN-derived peptides groups were similar in cell spreading. Osteogenic differentiation was observed in the peptide-modified Ti surface groups, compared with that of the noncoated Ti group. FN and GRGDSP/PHSRN coating enhanced the gene and protein expression of Runx2, osteocalcin, and bone sialoprotein. Alkaline phosphatase activity and matrix mineralization were also markedly enhanced in the Ti coated groups. CONCLUSION: The sandblasted Ti coated with FN or FN-derived peptides (GRGDSP/PHSRN) markedly enhance adhesion, proliferation, and differentiation of osteoblast-like cells compared with uncoated sandblasted Ti.