14-3-3ε protein-immobilized PCL-HA electrospun scaffolds with enhanced osteogenicity

Abstract

Adipose-derived mesenchymal stem cells (ASCs) accelerate the osteointegration of bone grafts and improve the efficiency in the formation of uniform bone tissue, providing a practical and clinically attractive approach in bone tissue regeneration. In this work, the effect of nanofibrous biomimetic matrices composed of poly(ε-caprolactone) (PCL), nanometric hydroxyapatite (nHA) particles and 14-3-3 protein isoform epsilon on the initial stages of human ASCs (hASCs) osteogenic differentiation was investigated. The cells were characterized by flow cytometry and induction to differentiation to adipogenic and osteogenic lineages. The isolated hASCs were induced to differentiate to osteoblasts over all scaffolds, and adhesion and viability of the hASCs were found to be similar. However, the activity of alkaline phosphatase (ALP) as early osteogenic marker in the PCL-nHA/protein scaffold was four times higher than in PCL-nHA and more than five times than the measured in neat PCL.

14-3-3ε protein-loaded 3D hydrogels favor osteogenesis

“…The results confirmed the expected positive effect of the protein on hASC proliferation. This strong effect of 14-3-3ε protein in hASC proliferation was previously observed on electrospun scaffolds modified with the same protein [6].…”

“…The strong effect of the 14-3-3ε recombinant protein on promoting hASCs osteogenic differentiation was evidenced when considering that the hydrogel+protein induced three times more ALP activity than did the neat hydrogels. In our previous work [ 6 ], the protein binding on the electrospun fiber surfaces enhanced the ALP activity in PCL-nHA/protein scaffolds which was four times higher than in non-modified PCL-nHA scaffolds, indicating osteogenic stimulation. The protein could be acting as a cytokine, enhancing both cell adhesion and proliferation, consistent with its presence in extracellular vesicles liberated from osteoblasts [ 17 ].…”

“…Viability was measured by alkaline phosphatase (ALP) activity assay [ 14 ]. Osteogenic differentiation of hASCs was induced with a drug cocktail containing 10 mM β-glycerophosphate, 0.1 µM dexamethasone and 50 µg/mL 2-phospho-L-ascorbic acid [ 6 ]. ALP activity was colorimetrically measured (multiskan FC, Thermo Fisher Scientific) by reading the absorption of p -nitrophenylphosphate at λ = 405 nm (ε = 18.3 mM −1 cm −1 ) on fixed (4% p- formaldehyde) and permeated cells (phosphate buffer saline, 0.05% Tween-20).…”

“…Incorporation of biochemical signals into the printed matrix is a convenient strategy to induce differentiation of stem cells [5]. Particularly, 14-3-3ε protein was recently studied for differentiation of human adipose derived stem cells (hASC) to osteoblasts on PCL-nHA scaffolds [6]. The 14-3-3 protein family includes in mammals seven highly conserved isoforms, involved in a wide range of vital regulatory processes.…”

“…The aim of this work was to develop printed scaffolds based on GelMA-alginate hydrogels encapsulating 14-3-3ε protein, which could stimulate the early stages of osteogenic differentiation of hASCs [6].…”