Spatial-Selective Volumetric 4D Printing and Single-Photon Grafting of Biomolecules within Centimeter-Scale Hydrogels via Tomographic Manufacturing.

Conventional additive manufacturing and biofabrication techniques are unable to edit the chemicophysical properties of the printed object postprinting. Herein, a new approach is presented, leveraging light-based volumetric printing as a tool to spatially pattern any biomolecule of interest in custom-designed geometries even across large, centimeter-scale hydrogels. As biomaterial platform, a gelatin norbornene resin is developed with tunable mechanical properties suitable for tissue engineering applications. The resin can be volumetrically printed within seconds at high resolution (23.68 ± 10.75 µm). Thiol-ene click chemistry allows on-demand photografting of thiolated compounds postprinting, from small to large (bio)molecules (e.g., fluorescent dyes or growth factors). These molecules are covalently attached into printed structures using volumetric light projections, forming 3D geometries with high spatiotemporal control and ≈50 µm resolution. As a proof of concept, vascular endothelial growth factor is locally photografted into a bioprinted construct and demonstrated region-dependent enhanced adhesion and network formation of endothelial cells. This technology paves the way toward the precise spatiotemporal biofunctionalization and modification of the chemical composition of (bio)printed constructs to better guide cell behavior, build bioactive cue gradients. Moreover, it opens future possibilities for 4D printing to mimic the dynamic changes in morphogen presentation natively experienced in biological tissues.

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.

Engineering microparticles based on solidified stem cell secretome with an augmented pro-angiogenic factor portfolio for therapeutic angiogenesis.

Tissue (re)vascularization strategies face various challenges, as therapeutic cells do not survive long enough in situ, while the administration of pro-angiogenic factors is hampered by fast clearance and insufficient ability to emulate complex spatiotemporal signaling. Here, we propose to address these limitations by engineering a functional biomaterial capable of capturing and concentrating the pro-angiogenic activities of mesenchymal stem cells (MSCs). In particular, dextran sulfate, a high molecular weight sulfated glucose polymer, supplemented to MSC cultures, interacts with MSC-derived extracellular matrix (ECM) components and facilitates their co-assembly and accumulation in the pericellular space. Upon decellularization, the resulting dextran sulfate-ECM hybrid material can be processed into MIcroparticles of SOlidified Secretome (MIPSOS). The insoluble format of MIPSOS protects protein components from degradation, while facilitating their sustained release. Proteomic analysis demonstrates that MIPSOS are highly enriched in pro-angiogenic factors, resulting in an enhanced pro-angiogenic bioactivity when compared to naïve MSC-derived ECM (cECM). Consequently, intravital microscopy of full-thickness skin wounds treated with MIPSOS demonstrates accelerated revascularization and healing, far superior to the therapeutic potential of cECM. Hence, the microparticle-based solidified stem cell secretome provides a promising platform to address major limitations of current therapeutic angiogenesis approaches.

Bioactive Decellularized Extracellular Matrix Derived from 3D Stem Cell Spheroids under Macromolecular Crowding Serves as a Scaffold for Tissue Engineering.

Scaffolds for tissue engineering aim to mimic the native extracellular matrix (ECM) that provides physical support and biochemical signals to modulate multiple cell behaviors. However, the majority of currently used biomaterials are oversimplified and therefore fail to provide a niche required for the stimulation of tissue regeneration. In the present study, 3D decellularized ECM (dECM) scaffolds derived from mesenchymal stem cell (MSC) spheroids and with intricate matrix composition are developed. Specifically, application of macromolecular crowding (MMC) to MSC spheroid cultures facilitate ECM assembly in a 3D configuration, resulting in the accumulation of ECM and associated bioactive components. Decellularized 3D dECM constructs produced under MMC are able to adequately preserve the microarchitecture of structural ECM components and are characterized by higher retention of growth factors. This results in a stronger proangiogenic bioactivity as compared to constructs produced under uncrowded conditions. These dECM scaffolds can be homogenously populated by endothelial cells, which direct the macroassembly of the structures into larger cell-carrying constructs. Application of empty scaffolds enhances intrinsic revascularization in vivo, indicating that the 3D dECM scaffolds represent optimal proangiogenic bioactive blocks for the construction of larger engineered tissue constructs.

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.

Macromolecular dextran sulfate facilitates extracellular matrix deposition by electrostatic interaction independent from a macromolecular crowding effect.

A faithful reconstruction of the native cellular microenvironment is instrumental for tissue engineering. Macromolecular crowding (MMC) empowers cells to deposit their own extracellular matrix (ECM) in greater amounts, and thus contributes to building tissue-specific complex microenvironments in vitro. Dextran sulfate (DxS, 500 kDa), a semi-synthetic sulfated polyglucose, was shown previously at a fractional volume occupancy (FVO) of 5.2% (v/v; 100 µg/ml) to act as a potent molecular crowding agent in vitro. When added to human mesenchymal stromal cell (MSC) cultures, DxS enhanced fibronectin and collagen I deposition several-fold also at concentrations with negligible FVO (<1% v/v). In a cell-free system, incubation of culture media supplemented with fetal bovine serum (FBS), purified fibronectin or collagen I with DxS led to a co-deposition of respective components, exhibiting a similar granular pattern as observed in cell culture. Aggregation of FBS components, fibronectin or collagen I with DxS was confirmed by dynamic light scattering, where an increase in hydrodynamic radius in the respective mixtures was observed. FBS- and fibronectin aggregates could be dissociated with increasing salt concentrations, indicating electrostatic forces to be responsible for the aggregation. Conversely, collagen I-DxS aggregates increased in size with increasing ion concentration, likely caused by charge screening of collagen I, which is net negatively charged at neutral pH, thus permitting weaker intermolecular interactions to occur. The incorporation of DxS into the ECM resulted in altered ECM topography and stiffness. DxS-supplemented cultures exhibited potentiated bioactivity, such as enhanced adipogenic and especially osteogenic differentiation under inductive conditions. We propose an alternative mechanism by which DxS drives ECM deposition via aggregation, and in an independent manner from MMC. A deeper understanding of the underlying mechanism will enable optimized engineering approaches for ECM-rich tissue constructs.

Cell-Derived Extracellular Matrix for Tissue Engineering and Regenerative Medicine.

Cell-derived extracellular matrices (CD-ECMs) captured increasing attention since the first studies in the 1980s. The biological resemblance of CD-ECMs to their in vivo counterparts and natural complexity provide them with a prevailing bioactivity. CD-ECMs offer the opportunity to produce microenvironments with costumizable biological and biophysical properties in a controlled setting. As a result, CD-ECMs can improve cellular functions such as stemness or be employed as a platform to study cellular niches in health and disease. Either on their own or integrated with other materials, CD-ECMs can also be utilized as biomaterials to engineer tissues de novo or facilitate endogenous healing and regeneration. This review provides a brief overview over the methodologies used to facilitate CD-ECM deposition and manufacturing. It explores the versatile uses of CD-ECM in fundamental research and therapeutic approaches, while highlighting innovative strategies. Furthermore, current challenges are identified and it is accentuated that advancements in methodologies, as well as innovative interdisciplinary approaches are needed to take CD-ECM-based research to the next level.

Rotary orbital suspension culture of embryonic stem cell-derived neural stem/progenitor cells: impact of hydrodynamic culture on aggregate yield, morphology and cell phenotype.

Embryonic stem (ES)-derived neural stem/progenitor cells (ES-NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single-cell suspensions of ES-NSPCs, to obtain homogeneously-sized ES-NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES-NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller-sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES-NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N-cadherin and ß-catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase-2 (MMP-2) activity and percentage of neuronal cells. In line with the increased MMP-2 activity levels found, hydrodynamically-cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single-cell suspensions of ES-NSPCs, besides allowing the formation of homogeneously-sized neurospheres, promoted ES-NSPC differentiation and outward migration, possibly by influencing the expression of cell-cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly-sized ES-NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or pharmacological screening. Copyright © 2016 John Wiley & Sons, Ltd.

Reduced glucosylceramide in the mouse model of Fabry disease: correction by successful enzyme replacement therapy.

Fabry disease is an X-linked lysosomal storage disease (LSD) caused by deficient activity of α-Galactosidase A (α-Gal A). As a result, glycosphingolipids, mainly globotriaosylceramide (Gb3), progressively accumulate in body fluids and tissues. Studies aiming at the identification of secondary lipid alterations in Fabry disease may be potentially useful for the monitorization of the response to enzyme replacement therapy (ERT) and development of future therapies. The focus of this study was to evaluate if α-Gal A deficiency has an effect on two key groups of molecules of sphingolipids metabolism: glucosylceramides (GlucCers) and ceramides (Cers). Studies performed in a mouse model of Fabry disease showed reduced level of GlucCer and normal level of Cer in plasma, liver, spleen, kidney and heart. Moreover, analysis of GlucCer isoforms in Fabry knockout mice showed that GlucCer isoforms are unequally reduced in different tissues of these animals. ERT had a specific effect on the liver's GlucCer levels of Fabry knockout mice, increasing hepatic GlucCer to the levels observed in wild type mice. In contrast to Fabry knockout mice, plasma of Fabry patients had normal GlucCer and Cer but an increased GlucCer/Cer ratio. This alteration showed a positive correlation with plasma globotriaosylsphingosine (lyso-Gb3) concentration. In conclusion, this work reveals novel secondary lipid imbalances caused by α-Gal A deficiency.