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Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Fudan University

Prostaglandin E2 has exhibited pleiotropic effects in the regulation of glomerulosclerosis progression through its four receptors. The current study aimed to evaluate the effect of prostaglandin receptor EP4 on mesangial cell proliferation. In vivo, 5/6 nephrectomy was introduced into EP4+/‑ and wild‑type (WT) mice. Clinical parameters were monitored post‑surgery. At 8 weeks post‑surgery, glomerular fibrosis‑associated indicators were measured by immunohistochemical staining and trichrome staining. In vitro, mesangial cells in different groups (transfected with green fluorescent protein, AD‑EF4 or AD‑CRE) were exposed to transforming growth factor (TGF)‑β1 for 24 h to detect the level of downstream signaling. Corresponding signaling inhibitors were also used to validate the signaling effects. Following surgery, EP4+/‑ mice presented a higher survival rate and normal urine volume compared with the WT group, and serum creatinine level and 24 h urine protein were lower in the EP4+/‑ mice. Furthermore, associated profibrotic indicators were identified to have decreased at 8 weeks post‑surgery along with less tubule‑interstitial fibrosis. In vivo, the inhibition of extracellular signal‑regulated kinase and P38 phosphorylation alleviated the accumulation of mesangial matrix, and these signals were enhanced when EP4 was overexpressed. EP4 enhancement aggravated imbalanced mesangial cell proliferation stimulated by TGF‑β1 and GS of mice treated with 5/6 nephrectomy through the Smad and mitogen‑activated protein kinase pathways.

EP4 knockdown alleviates glomerulosclerosis through Smad and MAPK pathways in mesangial cells.

Sepsis is a common and life-threatening systemic disorder, often leading to acute injury of multiple organs. Here, we show that remote ischemic preconditioning (rIPC), elicited by brief episodes of ischemia and reperfusion in femoral arteries, provides protective effects against sepsis-induced acute kidney injury (AKI).  Limb rIPC was conducted on mice  24 h before the onset of cecal ligation and puncture (CLP), and serum exosomes derived from rIPC mice were infused into CLP-challenged recipients. , we extracted and identified exosomes from differentiated C2C12 cells (myotubes) subjected to hypoxia and reoxygenation (H/R) preconditioning, and the exosomes were administered to lipopolysaccharide (LPS)-treated mouse tubular epithelial cells (mTECs) or intravenously injected into CLP-challenged miR-21 knockout mice for rescue experiments.  Limb rIPC protected polymicrobial septic mice from multiple organ dysfunction, systemic accumulation of inflammatory cytokines and accelerated parenchymal cell apoptosis through upregulation of miR-21 in a hypoxia-inducible factor 1α (HIF-1α)-dependent manner in the ischemic limbs of mice. However, in miR-21 knockout mice or mice that received HIF-1α siRNA injection into hind limb muscles, the organ protection conferred by limb rIPC was abolished. Mechanistically, we discovered that miR-21 was transported from preischemic limbs to remote organs via serum exosomes. In kidneys, the enhanced exosomal miR-21 derived from cultured myotubes with H/R or the serum of mice treated with rIPC integrated into renal tubular epithelial cells and then targeted the downstream PDCD4/NF-κB and PTEN/AKT pathways, exerting anti-inflammatory and anti-apoptotic effects and consequently attenuating sepsis-induced renal injury both  and .  This study demonstrates a critical role for exosomal miR-21 in renoprotection conferred by limb rIPC against sepsis and suggests that rIPC and exosomes might serve as the possible therapeutic strategies for sepsis-induced kidney injury.

Delayed Remote Ischemic Preconditioning ConfersRenoprotection against Septic Acute Kidney Injury via Exosomal miR-21.

One of the challenges for extrusion bioprinting using low-viscosity bioinks is the fast gravity-driven sedimentation of cells. Cells in a hydrogel bioink that features low viscosity tend to settle to the bottom of the bioink reservoir, and as such, their bioprintability is hindered by association with the inhomogeneous cellularized structures that are deposited. This is particularly true in cases where longer periods are required to print complex or larger tissue constructs. Increasing the bioink's viscosity efficiently retards sedimentation but gives rise to cell membranolysis or functional disruption due to increased shear stress on the cells during the extrusion process. Inspired by the rainbow cocktail, we report the development of a multilayered modification strategy for gelatin methacryloyl (GelMA) bioink to manipulate multiple liquid interfaces, providing interfacial retention to retard cell sedimentation in the bioink reservoir. Indeed, the interfacial tension in our layer-by-layer bioink system, characterized by the pendant drop method, was found to be exponentially higher than the sedimental pull (Δ = ∼10 N) of cells, indicating that the interfacial retention is crucial for preventing cell sedimentation across the adjacent layers. It was demonstrated that the encapsulated cells displayed better dispersibility in constructs bioprinted using the multilayered GelMA bioink system than that of pristine GelMA where the index of homogeneity of the cell distribution in the multilayered bioink was 4 times that of the latter.

Hydrogel Bioink with Multilayered Interfaces Improves Dispersibility of Encapsulated Cells in Extrusion Bioprinting.

Uncoupling protein 1 (UCP1) is predominantly found in brown adipose tissue mitochondria, and mediates energy dissipation to generate heat rather than ATP via functional mitochondrial uncoupling. However, little is known about its expression and function in kidney.

Tianyi Pan

Institutes of Biomedical Sciences and Department of Systems Biology for Medicine,
Shanghai Medical College,
Institutes of Biomedical Sciences and Department of Systems Biology for Medicine, Shanghai Medical College

Fudan University

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

Tianyi Pan

Institutes of Biomedical Sciences and Department of Systems Biology for Medicine,Shanghai Medical College,Institutes of Biomedical Sciences and Department of Systems Biology for Medicine, Shanghai Medical College

Fudan University

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

Institutes of Biomedical Sciences and Department of Systems Biology for Medicine,
Shanghai Medical College,
Institutes of Biomedical Sciences and Department of Systems Biology for Medicine, Shanghai Medical College