Single-Step 3D Bioprinting of Alginate-Collagen Type I Hydrogel Fiber Rings to Promote Angiogenic Network Formation.

In the advent of tissue engineering and regenerative medicine, the demand for innovative approaches to biofabricate complex vascular structures is increasing. We describe a single-step 3D bioprinting method leveraging Aspect Biosystems RX1 technology, which integrates the crosslinking step at a flow-focusing junction, to biofabricate immortalized adult rat brain endothelial cell (SV-ARBEC)-encapsulated alginate-collagen type I hydrogel rings. This single-step biofabrication process involves the strategic layer-by-layer assembly of hydrogel rings, encapsulating SV-ARBECs in a spatially controlled manner while optimizing access to media and nutrients. The spatial arrangement of the SV-ARBECs within the rings promotes spontaneous angiogenic network formation and the constrained deposition of cells within the hydrogel matrix facilitates tissue-like organized vascular-like network development. This approach provides a platform that can be adapted to many different endothelial cell types and leveraged to better understand the mechanisms driving angiogenesis and vascular-network formation in 3D bioprinted constructs supporting the development of more complex tissue and disease models for advancing drug discovery, tissue engineering, and regenerative medicine applications.

Differentiation of Adipose-Derived Stem Cells into Vascular Smooth Muscle Cells for Tissue Engineering Applications.

Synthetic grafts have been developed for vascular bypass surgery, however, the risks of thrombosis and neointimal hyperplasia still limit their use. Tissue engineering with the use of adipose-derived stem cells (ASCs) has shown promise in addressing these limitations. Here we further characterized and optimized the ASC differentiation into smooth muscle cells (VSMCs) induced by TGF-ß and BMP-4. TGF-ß and BMP-4 induced a time-dependent expression of SMC markers in ASC. Shortening the differentiation period from 7 to 4 days did not impair the functional property of contraction in these cells. Stability of the process was demonstrated by switching cells to regular growth media for up to 14 days. The role of IGFBP7, a downstream effector of TGF-ß, was also examined. Finally, topographic and surface patterning of a substrate is recognized as a powerful tool for regulating cell differentiation. Here we provide evidence that a non-woven PET structure does not affect the differentiation of ASC. Taken together, our results indicate that VSMCs differentiated from ASCs are a suitable candidate to populate a PET-based vascular scaffolds. By employing an autologous source of cells we provide a novel alternative to address major issues that reduces long-term patency of currently vascular grafts.

A novel human induced pluripotent stem cell blood-brain barrier model: Applicability to study antibody-triggered receptor-mediated transcytosis.

We have developed a renewable, scalable and transgene free human blood-brain barrier model, composed of brain endothelial cells (BECs), generated from human amniotic fluid derived induced pluripotent stem cells (AF-iPSC), which can also give rise to syngeneic neural cells of the neurovascular unit. These AF-iPSC-derived BECs (i-BEC) exhibited high transendothelial electrical resistance (up to 1500 Ω cm2) inducible by astrocyte-derived molecular cues and retinoic acid treatment, polarized expression of functional efflux transporters and receptor mediated transcytosis triggered by antibodies against specific receptors. In vitro human BBB models enable pre-clinical screening of central nervous system (CNS)-targeting drugs and are of particular importance for assessing species-specific/selective transport mechanisms. This i-BEC human BBB model discriminates species-selective antibody- mediated transcytosis mechanisms, is predictive of in vivo CNS exposure of rodent cross-reactive antibodies and can be implemented into pre-clinical CNS drug discovery and development processes.

IGFBP-4 anti-angiogenic and anti-tumorigenic effects are associated with anti-cathepsin B activity.

Insulin-like growth factor-binding protein 4 (IGFBP-4/IBP-4) has potent IGF-independent anti-angiogenic and antitumorigenic effects. In this study, we demonstrated that these activities are located in the IGFBP-4 C-terminal protein fragment (CIBP-4), a region containing a thyroglobulin type 1 (Tg1) domain. Proteins bearing Tg1 domains have been shown to inhibit cathepsins, lysosomal enzymes involved in basement membrane degradation and implicated in tumor invasion and angiogenesis. In our studies, CIBP-4 was shown to internalize and co-localize with lysosomal-like structures in both endothelial cells (ECs) and glioblastoma U87MG cells. CIBP-4 also inhibited both growth factor-induced EC tubulogenesis in Matrigel and the concomitant increases in intracellular cathepsin B (CatB) activity. In vitro assays confirmed CIBP-4 capacity to block recombinant CatB activity. Biodistribution analysis of intravenously injected CIBP-4-Cy5.5 in a glioblastoma tumor xenograft model indicated targeted accumulation of CIBP-4 in tumors. Most importantly, CIBP-4 reduced tumor growth in this animal model by 60%. Pleiotropic anti-angiogenic and anti-tumorigenic activities of CIBP-4 most likely underlie its observed therapeutic potential against glioblastoma.

A novel automated cell-seeding device for tissue engineering of tubular scaffolds: design and functional validation.

Obtaining an efficient, uniform and reproducible cell seeding of porous tubular scaffolds constitutes a major challenge for the successful development of tissue-engineered vascular grafts. In this study, a novel automated cell-seeding device utilizing direct cell deposition, patterning techniques and scaffold rotation was designed to improve the cell viability, uniformity and seeding efficiency of tubular constructs. Quantification methods and imaging techniques were used to evaluate these parameters on the luminal and abluminal sides of fibrous polymer scaffolds. With the automated seeding method, a high cell-seeding efficiency (~89%), viability (~85%) and uniformity (~85-92%) were achieved for both aortic smooth muscle cells (AoSMCs) and aortic endothelial cells (AoECs). The duration of the seeding process was < 8 min. Initial cell density, cell suspension in matrix-containing media, duration of seeding process and scaffold rotation were found to affect the seeding efficiency. After few days of culture, a uniform longitudinal and circumferential cell distribution was achieved without affecting cell viability. Both cell types were viable and spread along the fibres after 28 h and 6 days of static incubation. This new automated cell-seeding method for tubular scaffolds is efficient, reliable and meets all the requirements for clinical applicability.

Insulin-like growth factor binding protein 7 exhibits tumor suppressive and vessel stabilization properties in U87MG and T98G glioblastoma cell lines.

Insulin-like growth factor binding protein 7 (IGFBP7) is downregulated in several solid cancers. IGFBP7 has been proposed to act as a tumor suppressor gene through mechanisms involving senescence and apoptotic pathways. The tumor suppressor effect of IGFBP7 in glioblastoma multiforme (GBM) was examined in this study using two human GBM cell lines, U87MG and T98G. Exogenously applied IGFBP7 (20 and 100 nM) significantly reduced U87MG (~70 and ~75%, respectively) and T98G (~37 and ~50%, respectively) cell growth in soft agar. IGFBP7 stimulated senescence-associated ß-galactosidase in both U87MG and T98G cells without stimulating apoptosis (annexin V and propidium iodide staining, expression of SMARCB1 or BNIP3L and caspase cleavage) or affecting phosphorylation of p44/42 MAPK. The inhibitory effect of IGFBP7 on U87MG cell growth was further assessed in vivo using U87MG cells grafted on the chick chorioallantoic membrane. In this model, U87MG cells formed solid and highly vascularized tumors that were reduced in size (~40%) when treated with 500 nM IGFBP7 compared with control tumors. Vessels in IGFBP7-treated tumors were clustered, unevenly distributed and associated with higher number of α-SMA positive cells compared with those in untreated tumors. IGFBP7 induced both aortic smooth muscle cell (AoSMC) chemoattraction and endothelial cell (EC) transdifferentiation into a SM-like cell phenotype. U87MG conditioned media-induced IGFBP7 expression in ECs was significantly inhibited by the cross-talk/interaction with SMCs. This study indicates that IGFBP7 suppresses U87MG tumor cell growth, induces cell senescence and participates in tumor vessel stabilization by promoting SMC/pericyte recruitment and differentiation.