N-Cadherin Mimetic Peptide Nanofiber System Induces Chondrogenic Differentiation of Mesenchymal Stem Cells.

Cadherins are vital for cell-to-cell interactions during tissue growth, migration, and differentiation processes. Both biophysical and biochemical inputs are generated upon cell-to-cell adhesions, which determine the fate of the mesenchymal stem cells (MSCs). The effect of cadherin interactions on the MSC differentiation still remains elusive. Here we combined the N-Cadherin mimetic peptide (HAV-PA) with the self-assembling E-PA and the resultant N-cadherin mimetic peptide nanofibers promoted chondrogenic differentiation of MSCs in conjunction with chondrogenic factors as a synthetic extracellular matrix system. Self-assembly of the precursor peptide amphiphile molecules HAV-PA and E-PA enable the organization of HAV peptide residues in close proximity to the cell interaction site, forming a supramolecular N-cadherin-like system. These bioactive peptide nanofibers not only promoted viability and enhanced adhesion of MSCs but also augmented the expression of cartilage specific matrix components compared to the nonbioactive control nanofibers. Overall, the N-cadherin mimetic peptide nanofiber system facilitated MSC commitment into the chondrogenic lineage presenting an alternative bioactive platform for stem-cell-based cartilage regeneration.

Spatial Organization of Functional Groups on Bioactive Supramolecular Glycopeptide Nanofibers for Differentiation of Mesenchymal Stem Cells (MSCs) to Brown Adipogenesis.

Spatial organization of bioactive moieties in biological materials has significant impact on the function and efficiency of these systems. Here, we demonstrate the effect of spatial organization of functional groups including carboxylate, amine, and glucose functionalities by using self-assembled peptide amphiphile (PA) nanofibers as a bioactive scaffold. We show that presentation of bioactive groups on glycopeptide nanofibers affects mesenchymal stem cells (MSCs) in a distinct manner by means of adhesion, proliferation, and differentiation. Strikingly, when the glutamic acid is present in the glycopeptide backbone, the PA nanofibers specifically induced differentiation of MSCs into brown adipocytes in the absence of any differentiation medium as shown by lipid droplet accumulation and adipogenic gene marker expression analyses. This effect was not evident in the other glycopeptide nanofibers, which displayed the same functional groups but with different spatial organization. Brown adipocytes are attractive targets for obesity treatment and are found in trace amounts in adults, which also makes this specific glycopeptide nanofiber system an attractive tool to study molecular pathways of brown adipocyte formation.

Adipocyte-derived extracellular vesicles increase insulin secretion through transport of insulinotropic protein cargo.

Adipocyte-derived extracellular vesicles (AdEVs) are membranous nanoparticles that convey communication from adipose tissue to other organs. Here, to delineate their role as messengers with glucoregulatory nature, we paired fluorescence AdEV-tracing and SILAC-labeling with (phospho)proteomics, and revealed that AdEVs transfer functional insulinotropic protein cargo into pancreatic ß-cells. Upon transfer, AdEV proteins were subjects for phosphorylation, augmented insulinotropic GPCR/cAMP/PKA signaling by increasing total protein abundances and phosphosite dynamics, and ultimately enhanced 1st-phase glucose-stimulated insulin secretion (GSIS) in murine islets. Notably, insulinotropic effects were restricted to AdEVs isolated from obese and insulin resistant, but not lean mice, which was consistent with differential protein loads and AdEV luminal morphologies. Likewise, in vivo pre-treatment with AdEVs from obese but not lean mice amplified insulin secretion and glucose tolerance in mice. This data suggests that secreted AdEVs can inform pancreatic ß-cells about insulin resistance in adipose tissue in order to amplify GSIS in times of increased insulin demand.

Nanocomposite Bioinks Based on Agarose and 2D Nanosilicates with Tunable Flow Properties and Bioactivity for 3D Bioprinting.

Three-dimensional (3D) bioprinting enables the controlled fabrication of complex constructs for tissue engineering applications and has been actively explored in recent years. However, its progress has been limited by the existing difficulties in the development of bioinks with suitable biocompatibility and mechanical properties and at the same time adaptability to the process. Herein, we describe the engineering of a nanocomposite agarose bioink with tailored properties using 2D nanosilicate additives. The suitability of agarose for 3D bioprinting has been debated due to its bioinert nature and compatibility with extrusion-based bioprinting. Nanosilicates were used to tailor the flow behavior of agarose solutions, and detailed rheological characterization of different bioink formulations was performed to elucidate the effect of nanosilicates on the flow behavior and gelation of agarose bioinks. The proper selection of nanosilicate concentration resulted in extrusion 3D printed structures with high shape fidelity and structural integrity. Moreover, the influence of addition of nanosilicates on the bioactivity of agarose was studied, and nanocomposite bioinks showed significant improvement in metabolic activity of encapsulated cells. The bioactivity of the nanocomposite bioinks was also evaluated through a cell spreading assay; the charged surfaces of nanosilicates resulted in full spreading and elongation of fibroblasts, and the extent of change in morphology of cells was found to be directly correlated with the nanosilicate concentration. Our findings suggested that engineered agarose-nanosilicate bioinks can be exploited as a new generation of hydrogel bioinks for extrusion 3D bioprinting with tunable flow properties and bioactivity.

Nanosilicate embedded agarose hydrogels with improved bioactivity.

This paper reports the synthesis of nanocomposite agarose hydrogels with improved bioactivity with the incorporation of anisotropic 2D nanosilicates (Laponite) to promote cell binding, growth and proliferation. Rheological measurements showed that the incorporation of nanosilicates slightly increased the gelation temperature (Tgel). The use of higher nanosilicate content at the constant agarose concentration improved the mechanical properties of the gels. Due to the non-swelling nature of agarose, the addition of nanosilicates did not result in any remarkable change in the swelling properties of the agarose gels, while collapsed agarose nanofibers were observed with the incorporation of nanosilicates. EDX analysis confirmed the presence of the embedded nanosilicates in the gel matrix. The existence of physical interactions between nanosilicate and agarose was demonstrated by FTIR over the shifting of SiO stretching band to a lower frequency. The encapsulated NIH/3T3 fibroblast cells showed enhanced proliferation and spreading in the presence of nanosilicates.