Engineering a small diameter vascular graft with mechanical and biological properties comparable to living tissues remains challenging. Often, current devices lead to thrombosis and unsatisfactory long-term patency as a result of poor blood compatibility and a mismatch between the mechanical properties of the living tissue and the implanted biomaterial. Addressing all these requirements is essential to produce scaffolds able to survive throughout the life of the patient. For this purpose, we fabricated a novel three-layered vascular graft by combining electrospinning and braiding. Mirroring the structure of human blood vessels, the proposed device is composed of three layers: the intima, the media, and the adventitia. The intima and media layers were obtained by sequentially electrospinning silk fibroin (SF) and poly(L-lactide-co-ε-caprolactone), with ratios selected to match the mechanical properties of the native tissue. For the outer layer, the adventitia, SF yarns were braided on top of the electrospun tubes to create a structure able to withstand high pressures. Compliance, Young's modulus and deformability of the obtained scaffold were similar to that of human blood vessels. Additionally, cytocompatibility of the two layers, media and intima, was assessed in vitro by analysing cell metabolic activity and proliferation of endothelial cells and smooth muscle cells, respectively. Furthermore, heparin functionalization of the scaffolds led to improved anticoagulant properties upon incubation in whole blood. The obtained results indicate a potential application of the herewith designed three-layered construct as a vascular graft for small diameter blood vessel engineering.

Homeostasis of hematopoietic stem and progenitor cells (HSPC) is controlled by a combination of biochemical and biophysical environmental cues in the bone marrow (BM) niche, where a tight balance of quiescence and proliferation of HSPC is maintained. Specifically, alongside soluble factors and extracellular matrix (ECM) proteins, spatial confinement and ECM stiffness have been recognized to be critical for regulation of HSPC fate. Here we employ a modular, glycosaminoglycan (GAG)-based biohybrid hydrogel system to balance proliferation of human HSPC and maintenance of quiescent hematopoietic stem cells (HSC) through simultaneous regulation of exogenous biochemical and biophysical cues. Our results demonstrate that HSPC respond to increased spatial confinement with lowered proliferation and cell cycling, which results in higher frequency of quiescent LTC-IC (long-term culture initiating cells), while GAG-rich 3D environments further support maintenance of the cells.

Homeostasis of hematopoietic stem cells (HSC) in the mammalian bone marrow stem cell niche is regulated by signals of the local microenvironment. Besides juxtacrine, endocrine and metabolic cues, paracrine and autocrine signals are involved in controlling quiescence, proliferation and differentiation of HSC with strong implications on expansion and differentiation ex vivo as well as in vivo transplantation. Towards this aim, a cell culture analysis on a polymer microcavity carrier platform was combined with a partial least square analysis of a mechanistic model of cell proliferation. We could demonstrate the discrimination of specific autocrine and paracrine signals from soluble factors as stimulating and inhibitory effectors in hematopoietic stem and progenitor cell culture. From that we hypothesize autocrine signals to be predominantly involved in maintaining the quiescent state of HSC in single-cell niches and advocate our analysis platform as an unprecedented option for untangling convoluted signaling mechanisms in complex cell systems being it of juxtacrine, paracrine or autocrine origin.

Deciphering exogenous cues that determine stem cell fate decisions is a persisting challenge of cell biology and bioengineering. In an effort to unravel the role of spatial constraints in the cell-instructive characteristics of bone marrow microenvironments, murine hematopoietic stem and progenitor cells (HSPC) were exposed to fibronectin-coated microcavities in vitro. Microcavity sizes were chosen to allow for the inclusion of either individual or multiple cells. Repopulation experiments using lethally irradiated mice showed that the maintenance of functional HSPC in culture critically depends on cavity dimensions. Short-term repopulating hematopoietic stem cells (ST-HSC) were found to be best supported within single-cell sized compartments while long-term repopulating HSC (LT-HSC) were maintained within both cavity sizes. In sum, the reported data reveal spatial restriction to be a simple but powerful means for directing HSPC fate ex vivo.