cell-patterning-using-magnetic-archimedes-strategy

Cell Patterning Using Magnetic-Archimedes Strategy

Cell patterning, allowing precise control of cell positioning, presents a unique advantage in the study of cell behavior. In this protocol, a cell ...

Zhejiang University School of Medicine

State Key Laboratory of Transvascular Implantation Devices

Cardiovascular Key Laboratory of Zhejiang Province

Calcific aortic valve disease (CAVD) is a primary cause of cardiovascular mortality; however, its mechanisms are unknown. Currently, no effective pharmacotherapy is available for CAVD. Aldo-keto reductase family 1 member B (Akr1B1) has been identified as a potential therapeutic target for valve interstitial cell calcification. Herein, we hypothesized that inhibition of Akr1B1 can attenuate aortic valve calcification.

Aldo-keto reductase family 1 member B induces aortic valve calcification by activating hippo signaling in valvular interstitial cells.

An aging population and a rapid increase in the incidence of degenerative valve diseases have led to greater use of bioprosthetic heart valves (BHVs). The durability of glutaraldehyde cross-linked bioprostheses currently available for clinical use is poor due to calcification, coagulation, and degradation. Decellularization can partially reduce calcification by removal of xenogenic cells, but can also lead to thrombosis, which can be addressed by further surface modification. The natural sulfated polysaccharide ulvan possesses antithrombotic and anti-inflammatory properties, and can behave as a heparinoid to immobilize proteins through their heparin binding sites. VE-cadherin antibody and the Arg-Glu-Asp-Val (REDV) peptide can facilitate selective endothelial cell attachment, adhesion and proliferation. In this study, we functionalized decellularized porcine pericardium (DPP) with ulvan, REDV, and VE-cadherin antibody (U-R-VE). Ulvan was covalently modified to act as a protective coating and spacer for VE-cadherin antibody, and to immobilize REDV. In in vitro tests, we found that functionalization significantly and selectively promoted adhesion and growth of endothelial cells while reducing platelet adhesion, inflammation, and in vitro calcification of DPPs. In an in vivo subdermal implantation model, U-R-VE modified DPP exhibited greater endothelialization potential and biocompatibility compared with unmodified pericardium. Thus, U-R-VE modification provides a promising solution to the problem of preparing BHVs with enhanced endothelialization potential.

Ulvan mediated VE cadherin antibody and REDV peptide co-modification to improve endothelialization potential of bioprosthetic heart valves.

Heart valves have extraordinary fatigue resistance which beat ≈3 billion times in a lifetime. Bioprosthetic heart valves (BHVs) made from fixed heteroplasm that are incrementally used in heart valve replacement fail to sustain the expected durability due to thrombosis, poor endothelialization, inflammation, calcification, and especially mechanical damage induced biocompatibility change. No effective strategy has been reported to conserve the biological properties of BHV after long-term fatigue test. Here, a double-network tough hydrogel is introduced, which interpenetrate and anchor into the matrix of decellularized porcine pericardium (dCell-PP) to form robust and stable conformal coatings and reduce immunogenicity. The ionic crosslinked hyaluronic acid (HA) network mimics the glycocalyx on endothelium which improves antithrombosis and accelerates endothelialization; the chemical crosslinked hydrophilic polyacrylamide (PAAm) network further enhances antifouling properties and strengthens the shielding hydrogels and their interaction with dCell-PP. In vitro and rabbit ex vivo shunt assay demonstrate great hemocompatibility of polyacrylamide/HA hydrogel hybrid PP (P/H-PP). Cell experiments and rat subcutaneous implantation confirm satisfactory endothelialization, biocompatibility, and anticalcification properties. For hydrodynamic experiment, P/H-PP gains full mark at different flow conditions and sustains excellent biomechanical and biological properties after 200 000 000 cycles. P/H double-network hydrogel armoring dCell-PP is a promising progress to extend BHV durability for clinical implantation therapy.

Double-Network Hydrogel Armored Decellularized Porcine Pericardium as Durable Bioprosthetic Heart Valves.

Weak tissue adhesion remains a major challenge in clinical translation of microneedle patches. Mimicking the structural features of honeybee stingers, stiff polymeric microneedles with unidirectionally backward-facing barbs were fabricated and embedded into various elastomer films to produce self-interlocking microneedle patches. The spirality of the barbing pattern was adjusted to increase interlocking efficiency. In addition, the micro-bleeding caused by microneedle puncturing adhered the porous surface of the patch substrate to the target tissue via coagulation. In the demonstrative application of myocardial infarction treatment, the bioinspired microneedle patches firmly fixed on challenging beating hearts, significantly reduced cardiac wall stress and strain in the infarct, and maintained left ventricular function and morphology. In addition, the microneedle patch was minimally invasively implanted onto beating porcine heart in 10 minutes, free of sutures and adhesives. Therefore, the honeybee stinger-inspired microneedles could provide an adaptive and convenient means to implant patches for various medical applications. STATEMENT OF SIGNIFICANCE: Adhesion between tissue and microneedle patches with smooth microneedles is usually weak. We introduce a novel barbing method of fabricating unidirectionally backward facing barbs with controllable spirality on the microneedles on microneedle patches. The microneedle patches self-interlock on mechanically dynamic beating hearts, similar to honeybee stingers. The micro-bleeding and coagulation on the porous surface provide additional adhesion force. The microneedle patches attenuate left ventricular remodeling via mechanical support and are compatible with minimally invasive implantation.

A honeybee stinger-inspired self-interlocking microneedle patch and its application in myocardial infarction treatment.

Fibrotic cardiac diseases are characterized by myocardial fibrosis that results in maladaptive cardiac remodeling. Cardiac fibroblasts (CFs) are the main cell type responsible for fibrosis. In response to stress or injury, intrinsic CFs develop into myofibroblasts and produce excess extracellular matrix (ECM) proteins. Myofibroblasts are mechanosensitive cells that can detect changes in tissue stiffness and respond accordingly. Previous studies have revealed that some mechanical stimuli control fibroblast behaviors, including ECM formation, cell migration, and other phenotypic traits. Further, metabolic alteration is reported to regulate fibrotic signaling cascades, such as the transforming growth factor-β pathway and ECM deposition. However, the relationship between metabolic changes and mechanical stress during fibroblast-to-myofibroblast transition remains unclear. This review aims to elaborate on the crosstalk between mechanical stress and metabolic changes during the pathological transition of cardiac fibroblasts.

Mechanical stress regulates the mechanotransduction and metabolism of cardiac fibroblasts in fibrotic cardiac diseases.

Post-traumatic massive hemorrhage demands immediately available first-aid supplies with reduced operation time and good surgical compliance. In-situ crosslinking gels that are flexibly adapting to the wound shape have a promising potential, but it is still hard to achieve fast gelation, on-demand adhesion, and wide feasibility at the same time.

White-light crosslinkable milk protein bioadhesive with ultrafast gelation for first-aid wound treatment.

Tissue engineering raised a high requirement to control cell distribution in defined materials and structures. In "ink"-based bioprintings, such as 3D printing and photolithography, cells were associated with inks for spatial orientation; the conditions suitable for one ink are hard to apply on other inks, which increases the obstacle in their universalization. The Magneto-Archimedes effect based (Mag-Arch) strategy can modulate cell locomotion directly without impelling inks. In a paramagnetic medium, cells were repelled from high magnetic strength zones due to their innate diamagnetism, which is independent of substrate properties. However, Mag-Arch has not been developed into a powerful bioprinting strategy as its precision, complexity, and throughput are limited by magnetic field distribution. By controlling the paramagnetic reagent concentration in the medium and the gaps between magnets, which decide the cell repelling scope of magnets, we created simultaneously more than a hundred micrometer scale identical assemblies into designed patterns (such as alphabets) with single/multiple cell types. Cell patterning models for cell migration and immune cell adhesion studies were conveniently created by Mag-Arch. As a proof of concept, we patterned a tumor/endothelial coculture model within a covered microfluidic channel to mimic epithelial-mesenchymal transition (EMT) under shear stress in a cancer pathological environment, which gave a potential solution to pattern multiple cell types in a confined space without any premodification. Overall, our Mag-Arch patterning presents an alternative strategy for the biofabrication and biohybrid assembly of cells with biomaterials featured in controlled distribution and organization, which can be broadly employed in tissue engineering, regenerative medicine, and cell biology research.

Programing Cell Assembly via Ink-Free, Label-Free Magneto-Archimedes Based Strategy.

Single-nanoparticle detection has received tremendous interest due to its significance in fundamental physics and biological applications. Here, we demonstrate an optical nanofibre-enabled microfluidic sensor for the detection and sizing of nanoparticles. Benefitting from the strong evanescent field outside the nanofibre, a nanoparticle close to the nanofibre can scatter a portion of the field energy to the environment, resulting in a decrease in the transmitted intensity of the nanofibre. On the other hand, the narrow and shallow microfluidic channel provides a femtoliter-scale detection region, making nanoparticles flow through the detection region one by one. By real-time monitoring of the transmitted intensity of the nanofibre, the detection of a single polystyrene (PS) nanoparticle as small as 100 nm in diameter and exosomes in solution is realised. Based on a statistical analysis, the mean scattering signal is related to the size of the nanoparticle. Experimentally, a mixture of nanoparticles of different diameters (200, 500, and 1000 nm) in solution is identified. To demonstrate its potential in biological applications, high-throughput counting of yeasts using a pair of microchannels and dual-wavelength detection of fluorescently labelled nanoparticles are realised. We believe that the developed nanoparticle sensor holds great potential for the multiplexed and rapid sensing of diverse viruses.

An optical nanofibre-enabled on-chip single-nanoparticle sensor.

Metal ions participate in many metabolic processes in the human body, and their homeostasis is crucial for life. In cardiovascular diseases (CVDs), the equilibriums of metal ions are frequently interrupted, which are related to a variety of disturbances of physiological processes leading to abnormal cardiac functions. Exogenous supplement of metal ions has the potential to work as therapeutic strategies for the treatment of CVDs. Compared with other therapeutic drugs, metal ions possess broad availability, good stability and safety and diverse drug delivery strategies. The delivery strategies of metal ions are important to exert their therapeutic effects and reduce the potential toxic side effects for cardiovascular applications, which are also receiving increasing attention. Controllable local delivery strategies for metal ions based on various biomaterials are constantly being designed. In this review, we comprehensively summarized the positive roles of metal ions in the treatment of CVDs from three aspects: protecting cells from oxidative stress, inducing angiogenesis, and adjusting the functions of ion channels. In addition, we introduced the transferability of metal ions in vascular reconstruction and cardiac tissue repair, as well as the currently available engineered strategies for the precise delivery of metal ions, such as integrated with nanoparticles, hydrogels and scaffolds.

Exogeneous metal ions as therapeutic agents in cardiovascular disease and their delivery strategies.

Calcific aortic valve disease is a prevalent cardiovascular disease with no available drugs capable of effectively preventing its progression. Hence, an efficient drug delivery system could serve as a valuable tool in drug screening and potentially enhance therapeutic efficacy. However, due to the rapid blood flow rate associated with aortic valve stenosis and the lack of specific markers, achieving targeted drug delivery for calcific aortic valve disease has proved to be challenging. Here we find that protease-activated-receptor 2 (PAR2) expression is up-regulated on the plasma membrane of osteogenically differentiated valvular interstitial cells. Accordingly, we develop a magnetic nanocarrier functionalized with PAR2-targeting hexapeptide for dual-active targeting drug delivery. We show that the nanocarriers effectively deliver XCT790-an anti-calcification drug-to the calcified aortic valve under extra magnetic field navigation. We demonstrate that the nano-cargoes consequently inhibit the osteogenic differentiation of valvular interstitial cells, and alleviate aortic valve calcification and stenosis in a high-fat diet-fed low-density lipoprotein receptor-deficient (Ldlr) mouse model. This work combining PAR2- and magnetic-targeting presents an effective targeted drug delivery system for treating calcific aortic valve disease in a murine model, promising future clinical translation.

Article	Year
Aldo-keto reductase family 1 member B induces aortic valve calcification by activating hippo signaling in valvular interstitial cells. Journal of molecular and cellular cardiology\| PubMed ID: 33045251	2021
Ulvan mediated VE cadherin antibody and REDV peptide co-modification to improve endothelialization potential of bioprosthetic heart valves. Materials science & engineering. C, Materials for biological applications\| PubMed ID: 34474888	2021
Double-Network Hydrogel Armored Decellularized Porcine Pericardium as Durable Bioprosthetic Heart Valves. Advanced healthcare materials\| PubMed ID: 34969157	2022
A honeybee stinger-inspired self-interlocking microneedle patch and its application in myocardial infarction treatment. Acta biomaterialia\| PubMed ID: 36116725	2022
Mechanical stress regulates the mechanotransduction and metabolism of cardiac fibroblasts in fibrotic cardiac diseases. European journal of cell biology\| PubMed ID: 36696810	2023
White-light crosslinkable milk protein bioadhesive with ultrafast gelation for first-aid wound treatment. Biomaterials research\| PubMed ID: 36737833	2023
Programing Cell Assembly via Ink-Free, Label-Free Magneto-Archimedes Based Strategy. ACS nano\| PubMed ID: 37363813	2023
An optical nanofibre-enabled on-chip single-nanoparticle sensor. Lab on a chip\| PubMed ID: 37874569	2023
Exogeneous metal ions as therapeutic agents in cardiovascular disease and their delivery strategies. Regenerative biomaterials\| PubMed ID: 38173776	2024
Enhancing aortic valve drug delivery with PAR2-targeting magnetic nano-cargoes for calcification alleviation. Nature communications\| PubMed ID: 38228638	2024

Tanchen Ren

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