Stabilization and improved functionality of three-dimensional perfusable microvascular networks in microfluidic devices under macromolecular crowding.

BACKGROUND: There is great interest to engineer in vitro models that allow the study of complex biological processes of the microvasculature with high spatiotemporal resolution. Microfluidic systems are currently used to engineer microvasculature in vitro, which consists of perfusable microvascular networks (MVNs). These are formed through spontaneous vasculogenesis and exhibit the closest resemblance to physiological microvasculature. Unfortunately, under standard culture conditions and in the absence of co-culture with auxiliary cells as well as protease inhibitors, pure MVNs suffer from a short-lived stability. METHODS: Herein, we introduce a strategy for stabilization of MVNs through macromolecular crowding (MMC) based on a previously established mixture of Ficoll macromolecules. The biophysical principle of MMC is based on macromolecules occupying space, thus increasing the effective concentration of other components and thereby accelerating various biological processes, such as extracellular matrix deposition. We thus hypothesized that MMC will promote the accumulation of vascular ECM (basement membrane) components and lead to a stabilization of MVN with improved functionality. RESULTS: MMC promoted the enrichment of cellular junctions and basement membrane components, while reducing cellular contractility. The resulting advantageous balance of adhesive forces over cellular tension resulted in a significant stabilization of MVNs over time, as well as improved vascular barrier function, closely resembling that of in vivo microvasculature. CONCLUSION: Application of MMC to MVNs in microfluidic devices provides a reliable, flexible and versatile approach to stabilize engineered microvessels under simulated physiological conditions.

Dextran sulfate-amplified extracellular matrix deposition promotes osteogenic differentiation of mesenchymal stem cells.

The development of bone-like tissues in vitro that exhibit key features similar to those in vivo is needed to produce tissue models for drug screening and the study of bone physiology and disease pathogenesis. Extracellular matrix (ECM) is a predominant component of bone in vivo; however, as ECM assembly is sub-optimal in vitro, current bone tissue engineering approaches are limited by an imbalance in ECM-to-cell ratio. We amplified the deposition of osteoblastic ECM by supplementing dextran sulfate (DxS) into osteogenically induced cultures of human mesenchymal stem cells (MSCs). DxS, previously implicated to act as a macromolecular crowder, was recently demonstrated to aggregate and co-precipitate major ECM components, including collagen type I, thereby amplifying its deposition. This effect was re-confirmed for MSC cultures undergoing osteogenic induction, where DxS supplementation augmented collagen type I deposition, accompanied by extracellular osteocalcin accumulation. The resulting differentiated osteoblasts exhibited a more mature osteogenic gene expression profile, indicated by a strong upregulation of the intermediate and late osteogenic markers ALP and OCN, respectively. The associated cellular microenvironment was also enriched in bone morphogenetic protein 2 (BMP-2). Interestingly, the resulting decellularized matrices exhibited the strongest osteo-inductive effects on re-seeded MSCs, promoted cell proliferation, osteogenic marker expression and ECM calcification. Taken together, these findings suggest that DxS-mediated enhancement of osteogenic differentiation by MSCs is mediated by the amplified ECM, which is enriched in osteo-inductive factors. We have thus established a simple and reproducible approach to generate ECM-rich bone-like tissue in vitro with sequestration of osteo-inductive factors. STATEMENT OF SIGNIFICANCE: As extracellular matrix (ECM) assembly is significantly retarded in vitro, the imbalance in ECM-to-cell ratio hampers current in vitro bone tissue engineering approaches in their ability to faithfully resemble their in vivo counterpart. We addressed this limitation by leveraging a poly-electrolyte mediated co-assembly and amplified deposition of ECM during osteogenic differentiation of human mesenchymal stem cells (MSCs). The resulting pericelluar space in culture was enriched in organic and inorganic bone ECM components, as well as osteo-inductive factors, which promoted the differentiation of MSCs towards a more mature osteoblastic phenotype. These findings thus demonstrated a simple and reproducible approach to generate ECM-rich bone-like tissue in vitro with a closer recapitulation of the in vivo tissue niche.

Hyaluronic acid drives mesenchymal stromal cell-derived extracellular matrix assembly by promoting fibronectin fibrillogenesis.

Hyaluronic acid (HA)-based biomaterials have been demonstrated to promote wound healing and tissue regeneration, owing to the intrinsic and important role of HA in these processes. A deeper understanding of the biological functions of HA would enable better informed decisions on applications involving HA-based biomaterial design. HA and fibronectin are both major components of the provisional extracellular matrix (ECM) during wound healing and regeneration. Both biomacromolecules exhibit the same spatiotemporal distribution, with fibronectin possessing direct binding sites for HA. As HA is one of the first components present in the wound healing bed, we hypothesized that HA may be involved in the deposition, and subsequently fibrillogenesis, of fibronectin. This hypothesis was tested by exposing cultures of mesenchymal stromal cells (MSCs), which are thought to be involved in the early phase of wound healing, to high molecular weight HA (HMWHA). The results showed that treatment of human bone marrow derived MSCs (bmMSCs) with exogenous HMWHA increased fibronectin fibril formation during early ECM deposition. On the other hand, partial depletion of endogenous HA led to a drastic impairment of fibronectin fibril formation, despite detectable granular presence of fibronectin in the perinuclear region, comparable to observations made under the well-established ROCK inhibition-mediated impairment of fibronectin fibrillogenesis. These findings suggest the functional involvement of HA in effective fibronectin fibrillogenesis. The hypothesis was further supported by the co-alignment of fibronectin, HA and integrin α5 at sites of ongoing fibronectin fibrillogenesis, suggesting that HA might be directly involved in fibrillar adhesions. Given the essential function of fibronectin in ECM assembly and maturation, HA may play a major enabling role in initiating and propagating ECM deposition. Thus, HA, as a readily available biomaterial, presents practical advantages for de novo ECM-rich tissue formation in tissue engineering and regenerative medicine.