Cell Microencapsulation within Gelatin-PEG Microgels Using a Simple Pipet Tip-Based Device.

Microgels are microscale particles of hydrogel that can be laden with cells and used to create macroporous tissue constructs. Their ability to support cell-ECM and cell-cell interactions, along with the high levels of nutrient and metabolite exchange facilitated by their high surface area-to-volume ratio, means that they are attracting increasing attention for a variety of tissue regeneration applications. Here, we present methods for fabricating and modifying the structure of microfluidic devices using commonly available laboratory consumables including pipet tips and PTFE and silicon tubing to produce microgels. Different microfluidic devices realized the controlled generation of a wide size range (130-800 µm) of microgels for cell encapsulation. Subsequently, we describe the process of encapsulating mesenchymal stromal cells in microgels formed by photo-cross-linking of gelatin-norbornene and PEG dithiol. The introduced pipet-based chip offers simplicity, tunability, and versatility, making it easily assembled in most laboratories to effectively produce cell-laden microgels for various applications in tissue engineering.

Application of an integrated loach-plant-substrate-microbes non-aerated saturated vertical flow constructed wetlands: Mechanisms of pollutants removal and greenhouse gases reduction.

This study established an integrated loach-plant-substrate-microbes non-aerated saturated vertical flow constructed wetlands (VFCWs) to enhance pollutants removal efficiencies and reduce greenhouse gas emissions simultaneously. The results of the VFCWs experiment indicated that the removal efficiencies of chemical oxygen demand, total phosphorous, and total nitrogen in loach systems were significantly higher than those of non-loach systems, achieving 59.16%, 35.98%, and 40.96%, respectively. The CH4 and N2O emission fluxes were also significantly reduced in the integrated system, resulting in lower global warming potential (GWP) and GWP per unit of pollutants removal. Loaches promoted the transportation of oxygen, facilitated the re-contact and utilization of sediments, reduced CH4 emission, and enhanced nitrogen conversion and phosphorus accumulation. Increased bioavailable carbon and nitrate-nitrogen in the integrated system improved the abundance of denitrifying bacteria, which supported complete denitrification, reducing N2O emissions with high pollutant removal.

Elevated mir-145-5p is associated with skeletal muscle dysfunction and triggers apoptotic cell death in C2C12 myotubes.

Skeletal muscle dysfunction is a common comorbidity of chronic obstructive pulmonary disease (COPD), and the molecular mechanisms regarding to the pathogenesis of this disease have not been elucidated. In this study, a novel miR-145-5p was significantly upregulated in the serum collected from patients with COPD-associated muscle atrophy, in contrast with the normal participants. Then, we evidenced that silencing of miR-145-5p suppressed cell death and elongated cell survival during cell culture process. Consistently, upregulation of miR-145-5p induced cell apoptosis and restrain cell viability in the C2C12 cells, suggesting that miR-145-5p contributes to cell death. Further experiments evidenced that miR-145-5p decreased the expression levels of phosphorylated PI3K (p-PI3K), Akt (p-Akt) and mTOR (p-mTOR) to inactivate the PI3K/Akt/mTOR pathway, and this pathway was also reactivated by miR-145-5p ablation. Finally, we proved that the protective effects of miR-145-5p ablation were abrogated by co-treating cells with PI3K inhibitor LY294002. Taken together, we concluded that miR-145-5p promoted cell death to facilitate muscle dysfunctions via inactivating the PI3K/Akt/mTOR pathway.

Cell-laden injectable microgels: Current status and future prospects for cartilage regeneration.

Injectable hydrogels have been employed extensively as versatile materials for cartilage regeneration due to their excellent biocompatibility, tunable structure, and ability to accommodate bioactive factors, as well as their ability to be locally delivered via minimally invasive injection to fill irregular defects. More recently, in vitro and in vivo studies have revealed that processing these materials to produce cell-laden microgels can enhance cell-cell and cell-matrix interactions and boost nutrient and metabolite exchange. Moreover, these studies have demonstrated gene expression profiles and matrix regeneration that are superior compared to conventional injectable bulk hydrogels. As cell-laden microgels and their application in cartilage repair are moving closer to clinical translation, this review aims to present an overview of the recent developments in this field. Here we focus on the currently used biomaterials and crosslinking strategies, the innovative fabrication techniques being used for the production of microgels, the cell sources used, the signals used for induction of chondrogenic differentiation and the resultant biological responses, and the ability to create three-dimensional, functional cartilage tissues. In addition, this review also covers the current clinical approaches for repairing cartilage as well as specific challenges faced when attempting the regeneration of damaged cartilage tissue. New findings related to the macroporous nature of the structures formed by the assembled microgel building blocks and the novel use of microgels in 3D printing for cartilage tissue engineering are also highlighted. Finally, we outline the challenges and future opportunities for employing cell-laden microgels in clinical applications.

Cellular extrusion bioprinting improves kidney organoid reproducibility and conformation.

Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here, we apply extrusion-based three-dimensional cellular bioprinting to deliver rapid and high-throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate the relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, three-dimensional bioprinting enables precise manipulation of biophysical properties, including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitates the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production delivers improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.

Interplay of Hydrogel Composition and Geometry on Human Mesenchymal Stem Cell Osteogenesis.

Microgels are emerging as an outstanding platform for tissue regeneration because they overcome issues associated with conventional bulk/macroscopic hydrogels such as limited cell-cell contact and cell communication and low diffusion rates. Owing to the enhanced mass transfer and injectability via a minimally invasive procedure, these microgels are becoming a promising approach for bone regeneration applications. Nevertheless, there still remains a huge gap between the understanding of how the hydrogel matrix composition can influence cell response and overall tissue formation when switching from bulk formats to microgel format, which is often neglected or rarely studied. Here, we fabricated polyethylene glycol-based microgels and bulk hydrogels incorporating gelatin and hyaluronic acid (HA), either individually or together, and assessed the impact of both hydrogel composition and format upon the osteogenic differentiation of encapsulated human bone marrow-derived mesenchymal stem cells (hBMSCs). Osteogenesis was significantly greater in microgels than bulk hydrogels for both gelatin alone (Gel) and gelatin HA composite (Gel:HA) hydrogels, as determined by the expression of Runt-related transcription factor (Runx2) and alkaline phosphatase (ALP) genes and mineral deposition. Interestingly, Gel and Gel:HA hydrogels behaved differently between bulk and microgel format. In bulk format, overall osteogenic outcomes were better in Gel:HA hydrogels, but in microgel format, while the level of osteogenic gene expression was equivalent between both compositions, the degree of mineralization was reduced in Gel:HA microgels. Investigation into the affinity of hydroxyapatite for the different matrix compositions indicated that the decreased mineralization of Gel:HA microgels was likely due to a low affinity of hydroxyapatite to bind to HA and support mineral deposition, which has a greater impact on microgels than bulk hydrogels. Together, these findings suggest that both hydrogel composition and format can determine the success of tissue formation and that there is a complex interplay of these two factors on both cell behavior and matrix deposition. This has important implications for tissue engineering, showing that hydrogel composition and geometry must be evaluated together when optimizing conditions for cell differentiation and tissue formation.

Protective effects of P2X7R antagonist in sepsis-induced acute lung injury in mice via regulation of circ_0001679 and circ_0001212 and downstream Pln, Cdh2, and Nprl3 expression.

BACKGROUND: Sepsis induces pulmonary P2X7 receptor (P2X7 R) expression and P2X7 R-knockout reduced lung inflammation in mice. The present study investigated the expression of circular RNA (circRNA) and mRNA in sepsis-induced acute lung injury (ALI) treated with a P2X7 R antagonist. METHODS: Sepsis was induced by tracheal administration of lipopolysaccharide (LPS), and the mice were then divided into two groups: without [sepsis + dimethyl sulfoxide (DMSO)] or with P2X7 R antagonist treatment (sepsis + P2X7 A). Sham mice were administrated sterile normal saline. Serum levels of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, pathological changes, cell apoptosis and P2X7 R expression in lung were assessed, followed by RNA sequencing (RNA-seq) and bioinformatics analyses. A quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to validate circRNAs and mRNAs. RESULTS: Compared to the sham group, LPS-induced sepsis produced obvious pathological changes in lung tissue, as well as increased apoptotic lung cells, serum TNF-α and IL-1ß levels, and P2X7 R expression; P2X7 R antagonism significantly ameliorated these changes. RNA-seq identified many dysregulated circRNAs and mRNAs during sepsis, whereas this changed with P2X7 R antagonism. RT-qPCR confirmed that Mus musculus (mmu)_circ_0001679, mmu_circ_0001212, phospholamban (Pln), cadherin-2 (Cdh2) and nitrogen permease regulator 3-like (Nprl3) expression were significantly increased in the sepsis + DMSO group compared to that in the sham group but were decreased in the sepsis + P2X7 A group compared to that in the sepsis + DMSO group. The circRNA-microRNA-mRNA coexpression network indicated that mmu_circ_0001679 may regulate Nprl3 and that mmu_circ_0001212 may similarly regulate Pln, Cdh2 and Nprl3 as a competing endogenous RNA. CONCLUSIONS: P2X7 R antagonism attenuates sepsis-induced ALI by inhibiting dysregulated expression of circRNA (circ_0001679, circ_0001212) and mRNA (Pln, Cdh2 and Nprl3).

Microencapsulation improves chondrogenesis in vitro and cartilaginous matrix stability in vivo compared to bulk encapsulation.

The encapsulation of cells into microgels is attractive for applications in tissue regeneration. While cells are protected against shear stress during injection, the assembly of microgels after injection into a tissue defect also forms a macroporous scaffold that allows effective nutrient transport throughout the construct. However, in most of current strategies that form microgel-based macroporous scaffold or higher-order structures, cells are seeded during or post the assembly process and not microencapsulated in situ. The objective of this study is to investigate the chondrogenic phenotype of microencapsulated fetal chondrocytes in a biocompatible, assembled microgel system vs. bulk gels and to test the stability of the constructs in vivo. Here, we demonstrate that cell microencapsulation leads to increased expression of cartilage-specific genes in a TGF-ß1-dependent manner. This correlates, as shown by histological staining, with the ability of microencapsulated cells to deposit cartilaginous matrix after migrating to the surface of the microgels, while keeping a macroscopic granular morphology. Implantation of precultured scaffolds in a subcutaneous mouse model results in vessel infiltration in bulk gels but not in assembled microgels, suggesting a higher stability of the matrix produced by the cells in the assembled microgel constructs. The cells are able to remodel the microgels as demonstrated by the gradual disappearance of the granular structure in vivo. The biocompatible microencapsulation and microgel assembly system presented in this article therefore hold great promise as an injectable system for cartilage repair.

Visible Light Activation of Nucleophilic Thiol-X Addition via Thioether Bimane Photocleavage for Polymer Cross-Linking.

On-demand photo-uncaging of reactive thiols have been employed in engineering biomaterial scaffolds for regulation of cellular activities. A drawback of the current photo-uncaging chemistry is the utilization of high energy UV light or 2-photon laser light, which may be harmful to cells and cause undesired side reactions within the biological environment. We introduce an effective approach for the caging of thiol using monobromobimane, which can be removed under irradiation of light at λ = 420 nm and in the presence of electrophiles, such as acrylate, propiolate and maleimide, for trapping of the newly release thiol. This chemical approach can be used in visible light-induced polymer coupling and cross-linking for the preparation of cell-laden hydrogels.

Cartilage tissue formation through assembly of microgels containing mesenchymal stem cells.

Current clinical approaches to treat articular cartilage degeneration provide only a limited ability to regenerate tissue with long-term durability and functionality. In this application, injectable bulk hydrogels and microgels containing stem cells can provide a suitable environment for tissue regeneration. However insufficient cell-cell interactions, low differentiation efficiency and poor tissue adhesion hinder the formation of high-quality hyaline type cartilage. Here, we have designed a higher order tissue-like structure using injectable cell-laden microgels as the building blocks to achieve human bone marrow-derived mesenchymal stem cell (hBMSC) long-term maintenance and chondrogenesis. We have demonstrated that a 4-arm poly(ethylene glycol)-N-hydroxysuccinimide (NHS) crosslinker induces covalent bonding between the microgel building blocks as well as the surrounding tissue mimic. The crosslinking process assembles the microgels into a 3D construct and preserves the viability and cellular functions of the encapsulated hBMSCs. This assembled microgel construct encourages upregulation of chondrogenic markers in both gene and glycosaminoglycan (GAG) expression levels. In addition, the regenerated tissue in the assembled microgels stained positively with Alcian blue and Safranin O exhibiting unique hyaline-like cartilage features. Furthermore, the immunostaining showed a favourable distribution and significantly higher content of type II collagen in the assembled microgels when compared to both the bulk hydrogel and pellet cultures. Collectively, this tissue adhesive hBMSC-laden microgel construct provides potential clinical opportunities for articular cartilage repair and other applications in regenerative medicine. STATEMENT OF SIGNIFICANCE: A reliable approach to reconstruct durable and fully functional articular cartilage tissue is required for effective clinical therapies. Here, injectable hydrogels together with cell-based therapies offer new treatment strategies in cartilage repair. For effective cartilage regeneration, the injectable hydrogel system needs to be bonded to the surrounding tissue and at the same time needs to be sufficiently stable for prolonged chondrogenesis. In this work, we utilised injectable hBMSC-laden microgels as the building blocks to create an assembled construct via N-hydroxysuccinimide-amine coupling. This crosslinking process also allows for rapid bonding between the assembled microgels and a surrounding tissue mimic. The resultant assembled microgel-construct provides both a physically stable and biologically dynamic environment for hBMSC chondrogenesis, leading to the production of a mature hyaline type cartilage structure.

Mechanically-sensitive miRNAs bias human mesenchymal stem cell fate via mTOR signalling.

Mechanotransduction is a strong driver of mesenchymal stem cell (MSC) fate. In vitro, variations in matrix mechanics invoke changes in MSC proliferation, migration and differentiation. However, when incorporating MSCs within injectable, inherently soft hydrogels, this dominance over MSC response substantially limits our ability to couple the ease of application of hydrogels with efficiently directed MSC differentiation, especially in the case of bone generation. Here, we identify differential miRNA expression in response to varying hydrogel stiffness and RhoA activity. We show that modulation of miR-100-5p and miR-143-3p can be used to bias MSC fate and provide mechanistic insight by demonstrating convergence on mTOR signalling. By modulating these mechanosensitive miRNAs, we can enhance osteogenesis in a soft 3D hydrogel. The outcomes of this study provide new understanding of the mechanisms regulating MSC mechanotransduction and differentiation, but also a novel strategy with which to drive MSC fate and significantly impact MSC-based tissue-engineering applications.

Wavelength-Selective Coupling and Decoupling of Polymer Chains via Reversible [2 + 2] Photocycloaddition of Styrylpyrene for Construction of Cytocompatible Photodynamic Hydrogels.

Reversible photocycloaddition reactions have previously been employed in chemical cross-linking for the preparation of biomaterial scaffolds. However, the processes require activation by high-energy UV light, rendering them unsuitable for modification in biological environments. Here we demonstrate that the [2 + 2] photocycloaddition of styrylpyrene can be activated by visible light at λ = 400-500 nm, enabling rapid and effective conjugation and cross-linking of poly(ethylene glycol) (PEG) in water and under mild irradiation conditions (I = 20 mW cm-2). Notably, the reversion of the cycloaddition can be triggered by low-energy UV light at λ = 340 nm, which allows for efficient cleavage of the dimer adduct. Using this wavelength-gated reversible photochemical reaction we are able to prepare PEG hydrogels and modulate their mechanical properties in a bidirectional manner. We also demonstrate healing of the fractured hydrogel by external light triggers. Furthermore, we show that human mesenchymal stem cells can be encapsulated within the gels with high viability post encapsulation. This photochemical approach is therefore anticipated to be highly useful in studies of cell mechanotransduction, with relevance to disease progression and tissue regeneration.

Visible-light-mediated cleavage of polymer chains under physiological conditions via quinone photoreduction and trimethyl lock.

We introduce a click and visible-light triggered unclick approach via thio-bromo reaction and hydroquinone photoreduction/trimethyl lock cleavage for polymer modifications. Both reactions can be carried out in water and at ambient temperature, enabling preparation of bioorthogonal hydrogels for encapsulation and controlled release of various cells.

Photolabile Hydrogels Responsive to Broad Spectrum Visible Light for Selective Cell Release.

We introduce an efficient method for the preparation of photolabile polymer linkers to be used in the fabrication of bioorthogonal and photodegradable hydrogels. The versatility of this synthesis strategy allows for incorporation of a series of chromophores responsive to addressable wavelengths of UV and broad spectrum visible light. Consequently, selective release of different cell types from composite hydrogels by user-defined timing can be achieved by irradiating the materials with different wavelengths of light.

Microfluidic Encapsulation of Human Mesenchymal Stem Cells for Articular Cartilage Tissue Regeneration.

Stem cell injections for the treatment of articular cartilage damage are a promising approach to achieve tissue regeneration. However, this method is encumbered by high cell apoptosis rates, low retention in the cartilage lesion, and inefficient chondrogenesis. Here, we have used a facile, very low cost-based microfluidic technique to create visible light-cured microgels composed of gelatin norbornene (GelNB) and a poly(ethylene glycol) (PEG) cross-linker. In addition, we have demonstrated that the process enables the rapid in situ microencapsulation of human bone marrow-derived mesenchymal stem cells (hBMSCs) under biocompatible microfluidic-processing conditions for long-term maintenance. The hBMSCs exhibited an unusually high degree of chondrogenesis in the GelNB microgels with chondro-inductive media, specifically toward the hyaline cartilage structure, with significant upregulation in type II collagen expression compared to the bulk hydrogel and "gold standard" pellet culture. Overall, we have demonstrated that these protein-based microgels can be engineered as promising therapeutic candidates for articular cartilage regeneration, with additional potential to be used in a variety of other applications in regenerative medicine.

Versatile Bioorthogonal Hydrogel Platform by Catalyst-Free Visible Light Initiated Photodimerization of Anthracene.

Recent developments in photochemistry have introduced new methods to prepare hydrogels initiated by nonharmful light which is essential for encapsulation of cells and bioactive components. However, bioorthogonal photoclick reactions generally requires two components for cross-linking and, in many cases, the formation of a reactive intermediate that may cross-react with nucleophiles in biological media. Here we report the utilization of a visible light triggered dimerization of electron-rich anthracene for polymer cross-linking to form bulk hydrogels and microgels. Incorporation of gelatin within the hydrogel enhanced cell attachment and viability after 7 days of culture and spatiotemporal conjugation of a bioactive component using photochemical dimerization of anthracene was demonstrated. This work therefore introduces a simple yet powerful tool for light modulated bioorthogonal polymer cross-linking, which can be utilized in various bioengineering applications.

In situ-forming click-crosslinked gelatin based hydrogels for 3D culture of thymic epithelial cells.

Hydrogels prepared from naturally derived gelatin can provide a suitable environment for cell attachment and growth, making them favourable materials in tissue engineering. However, physically crosslinked gelatin hydrogels are not stable under physiological conditions while chemical crosslinking of gelatin by radical polymerization may be harmful to cells. In this study, we attached the norbornene functional group to gelatin, which was subsequently crosslinked with a polyethylene glycol (PEG) linker via the nitrile oxide-norbornene click reaction. The rapid crosslinking process allows the hydrogel to be formed within minutes of mixing the polymer solutions under physiological conditions, allowing the gels to be used as injectable materials. The hydrogels properties including mechanical strength, swelling and degradation, can be tuned by changing either the ratio of the reacting groups or the total concentration of the polymer precursors. Murine embryonic fibroblastic cells cultured in soft gels (2 wt% of gelatin and 1 wt% of PEG linker) demonstrated high cell viability as well as similar phenotypic profiles (PDGFRα and MTS15) to Matrigel cultures over 5 days. Thymic epithelial cell and fibroblast co-cultures produced epithelial colonies in these gels following 7 days incubation. These studies demonstrate that gelatin based hydrogels, prepared using "click" crosslinking, provide a robust cell culture platform with retained benefits of the gelatin material, and are therefore suitable for use in various tissue engineering applications.

Facile One-step Micropatterning Using Photodegradable Methacrylated Gelatin Hydrogels for Improved Cardiomyocyte Organization and Alignment.

Hydrogels are often employed as temporary platforms for cell proliferation and tissue organization in vitro. Researchers have incorporated photodegradable moieties into synthetic polymeric hydrogels as a means of achieving spatiotemporal control over material properties. In this study protein-based photodegradable hydrogels composed of methacrylated gelatin (GelMA) and a crosslinker containing o-nitrobenzyl ester groups have been developed. The hydrogels are able to degrade rapidly and specifically in response to UV light and can be photopatterned to a variety of shapes and dimensions in a one-step process. Micropatterned photodegradable hydrogels are shown to improve cell distribution, alignment and beating regularity of cultured neonatal rat cardiomyocytes. Overall this work introduces a new class of photodegradable hydrogel based on natural and biofunctional polymers as cell culture substrates for improving cellular organization and function.