Impact of the Reduction Time-Dependent Electrical Conductivity of Graphene Nanoplatelet-Coated Aligned Bombyx mori Silk Scaffolds on Electrically Stimulated Axonal Growth.

Graphene-based nanomaterials, renowned for their outstanding electrical conductivity, have been extensively studied as electroconductive biomaterials (ECBs) for electrically stimulated tissue regeneration. However, using eco-friendly reducing agents like l-ascorbic acid (l-Aa) can result in lower conductive properties in these ECBs, limiting their full potential for smooth charge transfer in living tissues. Moreover, creating a flexible biomaterial scaffold using these materials that accurately mimics a specific tissue microarchitecture, such as nerves, poses additional challenges. To address these issues, this study developed a microfibrous scaffold of Bombyx mori (Bm) silk fibroin uniformly coated with graphene nanoplatelets (GNPs) through a vacuum coating method. The scaffold's electrical conductivity was optimized by varying the reduction period using l-Aa. The research systematically investigated how different reduction periods impact scaffold properties, focusing on electrical conductivity and its significance on electrically stimulated axonal growth in PC12 cells. Results showed that a 48 h reduction significantly increased surface electrical conductivity by 100-1000 times compared to a shorter or no reduction process. l-Aa contributed to stabilizing the reduced GNPs, demonstrated by a slow degradation profile and sustained conductivity even after 60 days in a proteolytic environment. ß (III) tubulin immunostaining of PC12 cells on varied silk:GNP scaffolds under pulsed electrical stimulation (ES, 50 Hz frequency, 1 ms pulse width, and amplitudes of 100 and 300 mV/cm) demonstrates accelerated axonal growth on scaffolds exhibiting higher conductivity. This is supported by upregulated intracellular Ca2+ dynamics immediately after ES on the scaffolds with higher conductivity, subjected to a prolonged reduction period. The study showcases a sustainable reduction approach using l-Aa in combination with natural Bm silk fibroin to create a highly conductive, mechanically robust, and stable silk:GNP-based aligned fibrous scaffold. These scaffolds hold promise for functional regeneration in electrically excitable tissues such as nerves, cardiac tissue, and muscles.

2P-FLIM unveils time-dependent metabolic shifts during osteogenic differentiation with a key role of lactate to fuel osteogenesis via glutaminolysis identified.

BACKGROUND: Human mesenchymal stem cells (hMSCs) utilize discrete biosynthetic pathways to self-renew and differentiate into specific cell lineages, with undifferentiated hMSCs harbouring reliance on glycolysis and hMSCs differentiating towards an osteogenic phenotype relying on oxidative phosphorylation as an energy source. METHODS: In this study, the osteogenic differentiation of hMSCs was assessed and classified over 14 days using a non-invasive live-cell imaging modality-two-photon fluorescence lifetime imaging microscopy (2P-FLIM). This technique images and measures NADH fluorescence from which cellular metabolism is inferred. RESULTS: During osteogenesis, we observe a higher dependence on oxidative phosphorylation (OxPhos) for cellular energy, concomitant with an increased reliance on anabolic pathways. Guided by these non-invasive observations, we validated this metabolic profile using qPCR and extracellular metabolite analysis and observed a higher reliance on glutaminolysis in the earlier time points of osteogenic differentiation. Based on the results obtained, we sought to promote glutaminolysis further by using lactate, to improve the osteogenic potential of hMSCs. Higher levels of mineral deposition and osteogenic gene expression were achieved when treating hMSCs with lactate, in addition to an upregulation of lactate metabolism and transmembrane cellular lactate transporters. To further clarify the interplay between glutaminolysis and lactate metabolism in osteogenic differentiation, we blocked these pathways using BPTES and α-CHC respectively. A reduction in mineralization was found after treatment with BPTES and α-CHC, demonstrating the reliance of hMSC osteogenesis on glutaminolysis and lactate metabolism. CONCLUSION: In summary, we demonstrate that the osteogenic differentiation of hMSCs has a temporal metabolic profile and shift that is observed as early as day 3 of cell culture using 2P-FLIM. Furthermore, extracellular lactate is shown as an essential metabolite and metabolic fuel to ensure efficient osteogenic differentiation and as a signalling molecule to promote glutaminolysis. These findings have significant impact in the use of 2P-FLIM to discover potent approaches towards bone tissue engineering in vitro and in vivo by engaging directly with metabolite-driven osteogenesis.

Thou shall not heal: Overcoming the non-healing behaviour of diabetic foot ulcers by engineering the inflammatory microenvironment.

Diabetic foot ulcers (DFUs) are a devastating complication for diabetic patients that have debilitating effects and can ultimately lead to limb amputation. Healthy wounds progress through the phases of healing leading to tissue regeneration and restoration of the barrier function of the skin. In contrast, in diabetic patients dysregulation of these phases leads to chronic, non-healing wounds. In particular, unresolved inflammation in the DFU microenvironment has been identified as a key facet of chronic wounds in hyperglyceamic patients, as DFUs fail to progress beyond the inflammatory phase and towards resolution. Thus, control over and modulation of the inflammatory response is a promising therapeutic avenue for DFU treatment. This review discusses the current state-of-the art regarding control of the inflammatory response in the DFU microenvironment, with a specific focus on the development of biomaterials-based delivery strategies and their cargos to direct tissue regeneration in the DFU microenvironment.

A Workflow to Produce a Low-Cost In Vitro Platform for the Electric-Field Pacing of Cellularised 3D Porous Scaffolds.

Endogenous electrically mediated signaling is a key feature of most native tissues, the most notable examples being the nervous and the cardiac systems. Biomedical engineering often aims to harness and drive such activity in vitro, in bioreactors to study cell disease and differentiation, and often in three-dimensional (3D) formats with the help of biomaterials, with most of these approaches adopting scaffold-free self-assembling strategies to create 3D tissues. In essence, this is the casting of gels which self-assemble in response to factors such as temperature or pH and have capacity to harbor cells during this process without imparting toxicity. However, the use of materials that do not self-assemble but can support 3D encapsulation of cells (such as porous scaffolds) warrants consideration given the larger repertoire this would provide in terms of material physicochemical properties and microstructure. In this method and protocol paper, we detail and provide design codes and assembly instructions to cheaply create an electrical pacing bioreactor and a Rig for Stimulation of Sponge-like Scaffolds (R3S). This setup has also been engineered to simultaneously perform live optical imaging of the in vitro models. To showcase a pilot exploration of material physiochemistry (in this aspect material conductivity) and microstructure (isotropy versus anisotropy), we adopt isotropic and anisotropic porous scaffolds composed of collagen or poly(3,4-ethylene dioxythiophene):polystyrenesulfonate (PEDOT:PSS) for their contrasting conductivity properties yet similar in porosity and mechanical integrity. Electric field pacing of mouse C3H10 cells on anisotropic porous scaffolds placed in R3S led to increased metabolic activity and enhanced cell alignment. Furthermore, after 7 days electrical pacing drove C3H10 alignment regardless of material conductivity or anisotropy. This platform and its design, which we have shared, have wide suitability for the study of electrical pacing of cellularized scaffolds in 3D in vitro cultures.

Probing organoid metabolism using fluorescence lifetime imaging microscopy (FLIM): The next frontier of drug discovery and disease understanding.

Organoid models have been used to address important questions in developmental and cancer biology, tissue repair, advanced modelling of disease and therapies, among other bioengineering applications. Such 3D microenvironmental models can investigate the regulation of cell metabolism, and provide key insights into the mechanisms at the basis of cell growth, differentiation, communication, interactions with the environment and cell death. Their accessibility and complexity, based on 3D spatial and temporal heterogeneity, make organoids suitable for the application of novel, dynamic imaging microscopy methods, such as fluorescence lifetime imaging microscopy (FLIM) and related decay time-assessing readouts. Several biomarkers and assays have been proposed to study cell metabolism by FLIM in various organoid models. Herein, we present an expert-opinion discussion on the principles of FLIM and PLIM, instrumentation and data collection and analysis protocols, and general and emerging biosensor-based approaches, to highlight the pioneering work being performed in this field.

An assessment of the response of human MSCs to hydrostatic pressure in environments supportive of differential chondrogenesis.

Mechanical stimulation can modulate the chondrogenic differentiation of stem/progenitor cells and potentially benefit tissue engineering (TE) of functional articular cartilage (AC). Mechanical cues like hydrostatic pressure (HP) are often applied to cell-laden scaffolds, with little optimization of other key parameters (e.g. cell density, biomaterial properties) known to effect lineage commitment. In this study, we first sought to establish cell seeding densities and fibrin concentrations supportive of robust chondrogenesis of human mesenchymal stem cells (hMSCs). High cell densities (15*106 cells/ml) were more supportive of sGAG deposition on a per cell basis, while collagen deposition was higher at lower seeding densities (5*106 cells/ml). Employment of lower fibrin (2.5 %) concentration hydrogels supported more robust chondrogenesis of hMSCs, with higher collagen type II and lower collagen type X deposition compared to 5 % hydrogels. The application of HP to hMSCs maintained in identified chondro-inductive culture conditions had little effect on overall levels of cartilage-specific matrix production. However, if hMSCs were first temporally primed with TGF-ß3 before its withdrawal, they responded to HP by increased sGAG production. The response to HP in higher cell density cultures was also associated with a metabolic shift towards glycolysis, which has been linked with a mature chondrocyte-like phenotype. These results suggest that mechanical stimulation may not be necessary to engineer functional AC grafts using hMSCs if other culture conditions have been optimised. However, such bioreactor systems can potentially be employed to better understand how engineered tissues respond to mechanical loading in vivo once removed from in vitro culture environments.

Melt electrowriting of a biocompatible photo-crosslinkable poly(D,L-lactic acid)/poly(ε-caprolactone)-based material with tunable mechanical and functionalization properties.

The use of polymeric biomaterials to create tissue scaffolds using additive manufacturing techniques is a well-established practice, owing to the incredible rapidity and complexity in design that modern 3D printing methods can provide. One frontier approach is melt electrowriting (MEW), a technique that takes advantage of electrohydrodynamic phenomena to produce fibers on the scale of 10's of microns with designs capable of high resolution and accuracy. Poly(ε-caprolactone) (PCL) is a material that is commonly used in MEW due to its favorable thermal properties, high stability, and biocompatibility. However, one of the drawbacks of this material is that it lacks the necessary chemical groups which allow covalent crosslinking of additional elements onto its structure. Attempts to functionalise PCL structures therefore often rely on the functional units to be applied externally via coatings or integrally mixed elements. Both can be extremely useful depending on their applications, but can add extra difficulties into the use of the resulting structures. Coatings require careful design and application to prevent rapid degradation, while elements mixed into the polymer melt must deal with the possibilities of phase separation and changes to MEW properties of the unadulterated polymer. With this in mind, this study sought to imbibe functionality to MEW-printed scaffolds using the approach of adding functional units directly, via covalent bonding of functional groups to the polymer itself. To this end, this study employs a recently developed class of polymers called acrylate-endcapped urethane-based polymers (AUPs). The polymer backbone of the specific AUP used consists of a poly(D,L-lactic acid) (PDLLA)/PCL copolymer chain, which is functionalized with 6 acrylate groups, 3 at either end. Through blending of the AUP with PCL, various concentrations of this mixture were used with MEW to produce scaffolds that possessed acrylate groups on their surface. Using UV crosslinking, these groups were tagged with Fluorescein-o-Acrylate to verify that PDLLA/PCL AUP/PCL blends facilitate the direct covalent bonding of external agents directly onto the MEW material. Blending of the AUP with PCL increases the scaffold's stiffness and ultimate strength. Finally, blends were proven to be highly biocompatible, with cells attaching and proliferating readily at day 3 and 7 post seeding. Through this work, PDLLA/PCL AUP/PCL blends clearly demonstrated as a biocompatible material that can be processed using MEW to create functionalised tissue scaffolds.

Macrophage metabolic profile is altered by hydroxyapatite particle size.

Since the recent observation that immune cells undergo metabolic reprogramming upon activation, there has been immense research in this area to not only understand the basis of such changes, but also to exploit metabolic rewiring for therapeutic benefit. In a resting state, macrophages preferentially utilise oxidative phosphorylation to generate energy; however, in the presence of immune cell activators, glycolytic genes are upregulated, and energy is generated through glycolysis. This facilitates the rapid production of biosynthetic intermediates and a pro-inflammatory macrophage phenotype. While this is essential to mount responses to infectious agents, more evidence is accumulating linking dysregulated metabolism to inappropriate immune responses. Given that certain biomaterials are known to promote an inflammatory macrophage phenotype, this prompted us to investigate if biomaterial particulates can impact on macrophage metabolism. Using micron and nano sized hydroxyapatite (HA), we demonstrate for the first time that these biomaterials can indeed drive changes in metabolism, and that this occurs in a size-dependent manner. We show that micronHA, but not nanoHA, particles upregulate surrogate markets of glycolysis including the glucose transporter (GLUT1), hexokinase 2 (HK2), GAPDH, and PKM2. Furthermore, we demonstrate that micronHA alters mitochondrial morphology and promotes a bioenergetic shift to favour glycolysis. Finally, we demonstrate that glycolytic gene expression is dependent on particle uptake and that targeting glycolysis attenuates the pro-inflammatory profile of micronHA-treated macrophages. These results not only further our understanding of biomaterial-based macrophage activation, but also implicate immunometabolism as a new area for consideration in intelligent biomaterial design and therapeutic targeting. STATEMENT OF SIGNIFICANCE: Several recent studies have reported that immune cell activation occurs concurrently with metabolic reprogramming. Furthermore, metabolic reprogramming of innate immune cells plays a prominent role in determining cellular phenotype and function. In this study we demonstrate that hydroxyapatite particle size alters macrophage metabolism, in turn driving their functional phenotype. Specifically, the pro-inflammatory phenotype promoted by micron-sized HA-particles is accompanied by changes in mitochondrial dynamics and a bioenergetic shift favouring glycolysis. This effect is not seen with nano-HA particles and can be attenuated upon inhibition of glycolysis. This study therefore not only identifies immunometabolism as a useful tool for characterising the immune response to biomaterials, but also highlights immunometabolism as a targetable aspect of the host response for therapeutic benefit.

Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis.

Background: Thoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the myostatin (MSTN) gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an in vitro system for functional experiments we established three novel equine skeletal muscle cell lines reflecting the variation in phenotype associated with MSTN genotype (CC/II, CT/IN and TT/NN for SNP g.66493737C > T/SINE insertion 227 bp polymorphism). Primary equine skeletal muscle myoblasts, isolated from Thoroughbred horse gluteus medius, were conditionally immortalised and evaluated to determine whether cell phenotype and metabolic function were comparable to functional characteristics previously reported for ex vivo skeletal muscle isolated from Thoroughbred horses with each genotype. Results: Primary myoblasts conditionally immortalised with the temperature sensitive SV40TtsA58 lentivirus vector successfully proliferated and could revert to their primary cell phenotype and differentiate into multinucleated myotubes. Skeletal muscle fibre type, MSTN gene expression, mitochondrial abundance, and mitochondrial function of the three MSTN genotype cell lines, were consistent with equivalent characterisation of ex vivo skeletal muscle samples with these genotypes. Furthermore, addition of coenzyme Q10 (CoQ10) to the cell lines improved mitochondrial function, an observation consistent with ex vivo skeletal muscle samples with these genotypes following supplementation with CoQ10 in the diet. Conclusions: The observation that the phenotypic characteristics and metabolic function of the cells lines are equivalent to ex vivo skeletal muscle indicates that this in vitro system will enable efficient and cost-effective analyses of equine skeletal muscle for a range of different applications including understanding metabolic function, testing of nutritional supplements, drug test development and gene doping test development. In the multi-billion-euro international Thoroughbred horse industry research advances in the biological function of skeletal muscle are likely to have considerable impact. Furthermore, this novel genotype-specific system may be adapted and applied to human biomedicine to improve understanding of the effects of myostatin in human physiology and medicine.

MXene functionalized collagen biomaterials for cardiac tissue engineering driving iPSC-derived cardiomyocyte maturation.

Electroconductive biomaterials are gaining significant consideration for regeneration in tissues where electrical functionality is of crucial importance, such as myocardium, neural, musculoskeletal, and bone tissue. In this work, conductive biohybrid platforms were engineered by blending collagen type I and 2D MXene (Ti3C2Tx) and afterwards covalently crosslinking; to harness the biofunctionality of the protein component and the increased stiffness and enhanced electrical conductivity (matching and even surpassing native tissues) that two-dimensional titanium carbide provides. These MXene platforms were highly biocompatible and resulted in increased proliferation and cell spreading when seeded with fibroblasts. Conversely, they limited bacterial attachment (Staphylococcus aureus) and proliferation. When neonatal rat cardiomyocytes (nrCMs) were cultured on the substrates increased spreading and viability up to day 7 were studied when compared to control collagen substrates. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were seeded and stimulated using electric-field generation in a custom-made bioreactor. The combination of an electroconductive substrate with an external electrical field enhanced cell growth, and significantly increased cx43 expression. This in vitro study convincingly demonstrates the potential of this engineered conductive biohybrid platform for cardiac tissue regeneration.

Non-invasive classification of macrophage polarisation by 2P-FLIM and machine learning.

In this study, we utilise fluorescence lifetime imaging of NAD(P)H-based cellular autofluorescence as a non-invasive modality to classify two contrasting states of human macrophages by proxy of their governing metabolic state. Macrophages derived from human blood-circulating monocytes were polarised using established protocols and metabolically challenged using small molecules to validate their responding metabolic actions in extracellular acidification and oxygen consumption. Large field-of-view images of individual polarised macrophages were obtained using fluorescence lifetime imaging microscopy (FLIM). These were challenged in real time with small-molecule perturbations of metabolism during imaging. We uncovered FLIM parameters that are pronounced under the action of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), which strongly stratifies the phenotype of polarised human macrophages; however, this performance is impacted by donor variability when analysing the data at a single-cell level. The stratification and parameters emanating from a full field-of-view and single-cell FLIM approach serve as the basis for machine learning models. Applying a random forests model, we identify three strongly governing FLIM parameters, achieving an area under the receiver operating characteristics curve (ROC-AUC) value of 0.944 and out-of-bag (OBB) error rate of 16.67% when classifying human macrophages in a full field-of-view image. To conclude, 2P-FLIM with the integration of machine learning models is showed to be a powerful technique for analysis of both human macrophage metabolism and polarisation at full FoV and single-cell level.

A preclinical model of cutaneous melanoma based on reconstructed human epidermis.

Malignant melanoma is among the tumor entities with the highest increase of incidence worldwide. To elucidate melanoma progression and develop new effective therapies, rodent models are commonly used. While these do not adequately reflect human physiology, two-dimensional cell cultures lack crucial elements of the tumor microenvironment. To address this shortcoming, we have developed a melanoma skin equivalent based on an open-source epidermal model. Melanoma cell lines with different driver mutations were incorporated into these models forming distinguishable tumor aggregates within a stratified epidermis. Although barrier properties of the skin equivalents were not affected by incorporation of melanoma cells, their presence resulted in a higher metabolic activity indicated by an increased glucose consumption. Furthermore, we re-isolated single cells from the models to characterize the proliferation state within the respective model. The applicability of our model for tumor therapeutics was demonstrated by treatment with a commonly used v-raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor vemurafenib. This selective BRAF inhibitor successfully reduced tumor growth in the models harboring BRAF-mutated melanoma cells. Hence, our model is a promising tool to investigate melanoma development and as a preclinical model for drug discovery.

Distinct stromal and immune cell interactions shape the pathogenesis of rheumatoid and psoriatic arthritis.

OBJECTIVES: Immune and stromal cell communication is central in the pathogenesis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA), however, the nature of these interactions in the synovial pathology of the two pathotypes can differ. Identifying immune-stromal cell crosstalk at the site of inflammation in RA and PsA is challenging. This study creates the first global transcriptomic analysis of the RA and PsA inflamed joint and investigates immune-stromal cell interactions in the pathogenesis of synovial inflammation. METHODS: Single cell transcriptomic profiling of 178 000 synovial tissue cells from five patients with PsA and four patients with RA, importantly, without prior sorting of immune and stromal cells. This approach enabled the transcriptomic analysis of the intact synovial tissue and identification of immune and stromal cell interactions. State of the art data integration and annotation techniques identified and characterised 18 stromal and 14 immune cell clusters. RESULTS: Global transcriptomic analysis of synovial cell subsets identifies actively proliferating synovial T cells and indicates that due to differential λ and κ immunoglobulin light chain usage, synovial plasma cells are potentially not derived from the local memory B cell pool. Importantly, we report distinct fibroblast and endothelial cell transcriptomes indicating abundant subpopulations in RA and PsA characterised by differential transcription factor usage. Using receptor-ligand interactions and downstream target characterisation, we identify RA-specific synovial T cell-derived transforming growth factor (TGF)-ß and macrophage interleukin (IL)-1ß synergy in driving the transcriptional profile of FAPα+THY1+ invasive synovial fibroblasts, expanded in RA compared with PsA. In vitro characterisation of patient with RA synovial fibroblasts showed metabolic switch to glycolysis, increased adhesion intercellular adhesion molecules 1 expression and IL-6 secretion in response to combined TGF-ß and IL-1ß treatment. Disrupting specific immune and stromal cell interactions offers novel opportunities for targeted therapeutic intervention in RA and PsA.

Cholesterol crystals drive metabolic reprogramming and M1 macrophage polarisation in primary human macrophages.

BACKGROUND AND AIMS: Metabolic reprogramming of innate immune cells is emerging as a key player in the progression of a number of chronic diseases, including atherosclerosis, where high rates of glycolysis correlate with plaque instability. This study aimed to investigate if cholesterol crystals, which are key atherosclerosis-associated DAMPs (damage/danger-associated molecular patterns), alter immune cell metabolism and whether this, in turn, impacts on macrophage phenotype and function. METHODS AND RESULTS: Primary human macrophages were treated with cholesterol crystals and expression of M1 (CXCL9, CXCL10) and M2-associated (MRC1, CCL13) macrophage markers, alarmins, and inflammatory cytokines were assessed either by real-time PCR or ELISA. Cholesterol crystal-induced changes in glycolytic markers were determined using real-time PCR and western blotting, while changes in cellular respiration and mitochondrial dynamics were examined via Seahorse analysis, Fluorescence Lifetime Imaging Microscopy (FLIM) and confocal microscopy. Treatment of macrophages with cholesterol crystals upregulated mRNA levels of CXCL9 and CXCL10, while concomitantly downregulating expression of MRC1 and CCL13. Cholesterol crystal--treated macrophages also exhibited a significant shift in metabolism to favour glycolysis, accompanied by the expression of key glycolytic markers GLUT1, Hexokinase 2, HIF1α, GAPDH and PFKFB3. Furthermore, we show that these effects are mediated upstream by the glycolytic enzyme, PKM2, and that direct inhibition of glycolysis or PKM2 nuclear localisation leads to a significant reduction in cholesterol crystal-induced inflammatory readouts. CONCLUSIONS: This study not only provides further insight into how atherosclerosis-associated DAMPs impact on immune cell function, but also highlights metabolic reprogramming as a potential therapeutic target for cholesterol crystal-related inflammation.

Beyond Chemistry: Tailoring Stiffness and Microarchitecture to Engineer Highly Sensitive Biphasic Elastomeric Piezoresistive Sensors.

Carbon-based nanoparticles and conductive polymers are two classes of materials widely used in the production of three-dimensional (3D) piezoresistive sensors. One conductive polymer, poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) has excellent stability and conductivity yet is limited in its application as a sensor, often existing upon a base, limiting its performance and potential. Despite much progress in the field of materials chemistry and polymer synthesis, one aspect we consider worthy of exploration is the impact that microstructure and stiffness may have on the sensitivity of 3D sensors. In this study, we report a strategy for fabricating biphasic electroactive sponges (EAS) that combine 3D porous PEDOT:PSS scaffolds possessing either an isotropic or anisotropic microarchitecture, infused with insulating elastomeric fillers of varying stiffness. When characterizing the electromechanical behavior of these EAS, a higher stiffness yields a higher strain gauge factor, with values as high as 387 for an isotropic microarchitecture infused with a stiff elastomer. The approach we describe is cost-effective and extremely versatile, by which one can fabricate piezoresistive sensors with adaptable sensitivity ranges and excellent high strain gauge factor with the underlying microarchitecture and insulant stiffness dictating this performance.

Inside the Joint of Inflammatory Arthritis Patients: Handling and Processing of Synovial Tissue Biopsies for High Throughput Analysis.

Inflammatory arthritis is a chronic systemic autoimmune disease of unknown etiology, which affects the joints. If untreated, these diseases can have a detrimental effect on the patient's quality of life, leading to disabilities, and therefore, exhibit a significant socioeconomic impact and burden. While studies of immune cell populations in arthritis patient's peripheral blood have been informative regarding potential immune cell dysfunction and possible patient stratification, there are considerable limitations in identifying the early events that lead to synovial inflammation. The joint, as the site of inflammation and the local microenvironment, exhibit unique characteristics that contribute to disease pathogenesis. Understanding the contribution of immune and stromal cell interactions within the inflamed joint has been met with several technical challenges. Additionally, the limited availability of synovial tissue biopsies is a key incentive for the utilization of high-throughput techniques in order to maximize information gain. This review aims to provide an overview of key methods and novel techniques that are used in the handling, processing and analysis of synovial tissue biopsies and the potential synergy between these techniques. Herein, we describe the utilization of high dimensionality flow cytometric analysis, single cell RNA sequencing, ex vivo functional assays and non-intrusive metabolic characterization of synovial cells on a single cell level based on fluorescent lifetime imaging microscopy. Additionally, we recommend important points of consideration regarding the effect of different storage and handling techniques on downstream analysis of synovial tissue samples. The introduction of new powerful techniques in the study of synovial tissue inflammation, brings new challenges but importantly, significant opportunities. Implementation of novel approaches will accelerate our path toward understanding of the mechanisms involved in the pathogenesis of inflammatory arthritis and lead to the identification of new avenues of therapeutic intervention.

The Trypanosoma brucei-Derived Ketoacids, Indole Pyruvate and Hydroxyphenylpyruvate, Induce HO-1 Expression and Suppress Inflammatory Responses in Human Dendritic Cells.

The extracellular parasite and causative agent of African sleeping sickness Trypanosoma brucei (T. brucei) has evolved a number of strategies to avoid immune detection in the host. One recently described mechanism involves the conversion of host-derived amino acids to aromatic ketoacids, which are detected at relatively high concentrations in the bloodstream of infected individuals. These ketoacids have been shown to directly suppress inflammatory responses in murine immune cells, as well as acting as potent inducers of the stress response enzyme, heme oxygenase 1 (HO-1), which has proven anti-inflammatory properties. The aim of this study was to investigate the immunomodulatory properties of the T. brucei-derived ketoacids in primary human immune cells and further examine their potential as a therapy for inflammatory diseases. We report that the T. brucei-derived ketoacids, indole pyruvate (IP) and hydroxyphenylpyruvate (HPP), induce HO-1 expression through Nrf2 activation in human dendritic cells (DC). They also limit DC maturation and suppress the production of pro-inflammatory cytokines, which, in turn, leads to a reduced capacity to differentiate adaptive CD4+ T cells. Furthermore, the ketoacids are capable of modulating DC cellular metabolism and suppressing the inflammatory profile of cells isolated from patients with inflammatory bowel disease. This study therefore not only provides further evidence of the immune-evasion mechanisms employed by T. brucei, but also supports further exploration of this new class of HO-1 inducers as potential therapeutics for the treatment of inflammatory conditions.

Loss of balance between protective and pro-inflammatory synovial tissue T-cell polyfunctionality predates clinical onset of rheumatoid arthritis.

OBJECTIVES: This study investigates pathogenic and protective polyfunctional T-cell responses in patient with rheumatoid arthritis (RA), individuals at risk (IAR) and healthy control (HC) synovial-tissue biopsies and identifies the presence of a novel population of pathogenic polyfunctional T-cells that are enriched in the RA joint prior to the development of clinical inflammation. METHODS: Pathway enrichment analysis of previously obtained RNAseq data of synovial biopsies from RA (n=118), IAR (n=20) and HC (n=44) was performed. Single-cell synovial tissue suspensions from RA (n=10), IAR (n=7) and HC (n=7) and paired peripheral blood mononuclear cells (PBMC) were stimulated in vitro and polyfunctional synovial T-cell subsets examined by flow cytometric analysis, simplified presentation of incredibly complex evaluations (SPICE) and FlowSom clustering. Flow-imaging was utilised to confirm specific T-cell cluster identification. Fluorescent lifetime imaging microscopy (FLIM) was used to visualise metabolic status of sorted T-cell populations. RESULTS: Increased plasticity of Tfh cells and CD4 T-cell polyfunctionality with enriched memory Treg cell responses was demonstrated in RA patient synovial tissue. Synovial-tissue RNAseq analysis reveals that enrichment in T-cell activation and differentiation pathways pre-dates the onset of RA. Switch from potentially protective IL-4 and granulocyte macrophage colony stimulating factor (GMCSF) dominated polyfunctional CD4 T-cell responses towards pathogenic polyfunctionality is evident in patient with IAR and RA synovial tissue. Cluster analysis reveals the accumulation of highly polyfunctional CD4+ CD8dim T-cells in IAR and RA but not HC synovial tissue. CD4+ CD8dim T-cells show increased utilisation of oxidative phosphorylation, a characteristic of metabolically primed memory T-cells. Frequency of synovial CD4+ CD8dim T-cells correlates with RA disease activity. CONCLUSION: Switch from potentially protective to pathogenic T-cell polyfunctionality pre-dates the onset of clinical inflammation and constitutes an opportunity for therapeutic intervention in RA.

Resident Macrophages and Their Potential in Cardiac Tissue Engineering.

Many facets of tissue engineered models aim at understanding cellular mechanisms to recapitulate in vivo behavior, to study and mimic diseases for drug interventions, and to provide a better understanding toward improving regenerative medicine. Recent and rapid advances in stem cell biology, material science and engineering, have made the generation of complex engineered tissues much more attainable. One such tissue, human myocardium, is extremely intricate, with a number of different cell types. Recent studies have unraveled cardiac resident macrophages as a critical mediator for normal cardiac function. Macrophages within the heart exert phagocytosis and efferocytosis, facilitate electrical conduction, promote regeneration, and remove cardiac exophers to maintain homeostasis. These findings underpin the rationale of introducing macrophages to engineered heart tissue (EHT), to more aptly capitulate in vivo physiology. Despite the lack of studies using cardiac macrophages in vitro, there is enough evidence to accept that they will be key to making EHTs more physiologically relevant. In this review, we explore the rationale and feasibility of using macrophages as an additional cell source in engineered cardiac tissues. Impact statement Macrophages play a critical role in cardiac homeostasis and in disease. Over the past decade, we have come to understand the many vital roles played by cardiac resident macrophages in the heart, including immunosurveillance, regeneration, electrical conduction, and elimination of exophers. There is a need to improve our understanding of the resident macrophage population in the heart in vitro, to better recapitulate the myocardium through tissue engineered models. However, obtaining them in vitro remains a challenge. Here, we discuss the importance of cardiac resident macrophages and potential ways to obtain cardiac resident macrophages in vitro. Finally, we critically discuss their potential in realizing impactful in vitro models of cardiac tissue and their impact in the field.